Anti-human CD52 immunoglobulins

ABSTRACT

The present invention relates to humanized immunoglobulins, mouse monoclonal antibodies and chimeric antibodies that have binding specificity for human CD52. The present invention further relates to a humanized immunoglobulin light chain and a humanized immunoglobulin heavy chain. The invention also relates to isolated nucleic acids, recombinant vectors and host cells that comprise a sequence which encodes a humanized immunoglobulin or immunoglobulin light chain or heavy chain, and to a method of preparing a humanized immunoglobulin. The humanized immunoglobulins can be used in therapeutic applications to treat, for example, autoimmune disease, cancer, non-Hodgkin&#39;s lymphoma, multiple sclerosis and chronic lymphocytic leukemia.

This application is a national stage application under 35 U.S.C. §371 ofInternational Application PCT/US2010/034704 (now pending), filed May 13,2010, which claims priority from U.S. Provisional Application61/177,837, filed May 13, 2009. The contents of the foregoing priorityapplications are incorporated by reference herein in their entirety.

A Sequence Listing associated with this application is being submittedelectronically via EFS-Web in text format, and is hereby incorporated byreference in its entirety into the specification. The name of the textfile containing the Sequence Listing is001662_(—)0029_(—)301_Sequence_Listing.txt. The text file, created onNov. 10, 2011, is 182,313 bytes in size.

BACKGROUND OF THE INVENTION

CD52 is a glycosylated, glycosylphosphatidylinositol (GPI)-anchored cellsurface protein found in abundance (500,000 molecules/cell) on a varietyof normal and malignant lymphoid cells (e.g., T and B cells). See, e.g.,Hale et al., J Biol regul Homeost Agents 15:386-391 (2001); Huh et al.,Blood 92: Abstract 4199 (1998); Elsner et al., Blood 88:4684-4693(1996); Gilleece et al., Blood 82:807-812 (1993); Rodig et al., ClinCancer Res 12:7174-7179 (2006); Ginaldi et al., Leuk Res 22:185-191(1998). CD52 is expressed at lower levels on myeloid cells such asmonocytes, macrophages, and dendritic cells, with little expressionfound on mature natural killer (NK) cells, neutrophils, andhematological stem cells. Id. CD52 is also produced by epithelial cellsin the epididymis and duct deferens, and is acquired by sperm duringpassage through the genital tract (Hale et al., 2001, supra; Domagala etal., Med Sci Monit 7:325-331 (2001)). The exact biological function ofCD52 remains unclear but some evidence suggests that it may be involvedin T cell migration and co-stimulation (Rowan et al., Int Immunol7:69-77 (1995); Masuyama et al., J Exp Med 189:979-989 (1999); Watanabeet al., Clin Immunol 120:247-259 (2006)). Campath-1H® (alemtuzumab,Campath®, MabCampath®) is a humanized anti-human CD52 monoclonalantibody that exhibits potent in vitro cytotoxic effects(antibody-dependent cell mediated cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC)).

Campath® recognizes an epitope which consists of the carboxy terminalfour amino acids of the mature CD52 protein and a portion of thenegatively charged GPI anchor. Due to its significant cytotoxic effects,Campath® is capable of depleting CD52 positive cells in vivo and it isapproved for front line and third line treatment of chronic lymphocyticleukemia (CLL). Campath® has been evaluated for its utility in thetreatment of several autoimmune diseases, including rheumatoidarthritis, vasculitis, myositis and Wegener's disease. However, the mostadvanced studies of Campath® are in treating relapsing remittingmultiple sclerosis (MS). These studies showed a significant improvementin time to relapse relative to an active comparator (Rebif® (i.e.,interferon beta-1a)).

A need exists for additional therapeutic agents and approaches to targetCD52.

SUMMARY OF THE INVENTION

Humanized Immunoglobulins

The invention relates to humanized immunoglobulins that have bindingspecificity for human CD52 (huCD52). They may comprise thecomplementarity determining regions (CDRs) of mouse anti-human CD52antibodies. The humanized immunoglobulins of the invention have aminoacid sequences that are different from other humanized immunoglobulins,and in particular from other humanized immunoglobulins that compriseCDRs of murine anti-human CD52 antibodies. The humanized immunoglobulinsof the invention are different from the humanized immunoglobulinCampath®. In some embodiments, they provide advantages over humanizedantibodies that comprise the CDRs of Campath®.

The humanized immunoglobulins described herein can comprise a humanizedheavy chain and a humanized light chain. In one embodiment, thehumanized immunoglobulin comprises a light chain comprising one or moreCDRs (e.g., all three CDRs) of SEQ ID NO: 3 and a heavy chain comprisingone or more CDRs (e.g., all three CDRs) of SEQ ID NO: 16; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 4 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 17; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 5 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 18; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 6 and a heavy chain comprising oneor more CDRs of SEQ ID NO: 19; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 7 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 20; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 8 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 21; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 9 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 22; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 10 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 23; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 11 anda heavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQID NO: 24; a light chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 12 and a heavy chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 25; a light chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 12 and a heavy chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 137; ora light chain comprising one or more CDRs (e.g., all three CDRs) of SEQID NO: 13 and a heavy chain sequence comprising one or more CDRs (e.g.,all three CDRs) of SEQ ID NO: 26. The CDRs in the above-mentioned SEQ IDNOs are indicated by FIGS. 2 and 3 and are referred to in Tables 1-6 asprovided herein.

In another embodiment, the humanized immunoglobulin that has bindingspecificity for human CD52 comprises a light chain comprising one ormore CDRs (e.g., all three) selected from the group consisting of SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ IDNO: 46, SEQ ID NO: 47, and SEQ ID NO: 48; a heavy chain comprising oneor more CDRs (e.g., all three) selected from the group consisting of SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ IDNO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72,SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 294; or such a light chainand such a heavy chain; wherein the humanized immunoglobulin is notCampath®.

In another embodiment, the humanized immunoglobulin that has a bindingspecificity for human CD52 comprises a light chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13; a heavy chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, or SEQ ID NO: 137; or such a light chain and such a heavy chain;wherein the humanized immunoglobulin is not Campath®.

In some embodiments, the framework region of the humanizedimmunoglobulin has at least 50% homology to the framework region of theimmunoglobulin from which the light chain CDRs and the heavy chain CDRsare obtained. For example, the framework region of the humanizedimmunoglobulin can be at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or even100% identical, to a germline human immunoglobulin sequence. In oneembodiment, the framework region of the humanized immunoglobulin can beobtained or derived from an IgG human antibody variable region. Inanother embodiment the CD52 is wildtype human CD52. In yet anotherembodiment, the humanized immunoglobulin can compete with alemtuzumabfor binding to human CD52, e.g., it can bind to an epitope that isidentical to, or which overlaps with, the epitope to which alemtuzumabbinds.

The invention also relates to a humanized light chain of a humanizedimmunoglobulin of the invention. In one embodiment, the humanized lightchain comprises one or more CDRs selected from the group consisting ofSEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48 or a combinationthereof, wherein the humanized light chain is not the humanized lightchain of Campath®.

In other embodiment, the humanized light chain comprises one or moreCDRs (e.g., all three CDRs) of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, wherein the humanizedlight chain is not the humanized light chain of Campath®.

The invention also relates to a humanized heavy chain of a humanizedimmunoglobulin of the invention. In one embodiment, the humanized heavychain comprises one or more CDRs of an Ig variable domain selected fromthe group consisting of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ IDNO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO:294, or a combination thereof, wherein the humanized heavy chain is notthe humanized heavy chain of Campath®.

In other embodiments, the humanized heavy chain comprises one or moreCDRs (e.g., all three CDRs) of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 137,wherein the humanized heavy chain is not the humanized heavy chain ofCampath®.

Preferably, the humanized immunoglobulins of the present inventioncomprise both a humanized light chain of the invention and a humanizedheavy chain of the invention.

In other embodiments, the invention provides a humanized immunoglobulinwhich binds to the same epitope on human CD52 as, or competes orcross-competes with, a mouse monoclonal antibody comprising a lightchain variable region of SEQ ID NO: 3 and a heavy chain variable regionof SEQ ID NO: 16; a light chain variable region of SEQ ID NO: 4 and aheavy chain variable region of SEQ ID NO: 17; a light chain variableregion of SEQ ID NO: 5 and a heavy chain variable region of SEQ ID NO:18; a light chain variable region of SEQ ID NO: 6 and a heavy chainvariable region of SEQ ID NO: 19; a light chain variable region of SEQID NO: 7 and a heavy chain variable region of SEQ ID NO: 20; a lightchain variable region of SEQ ID NO: 8 and a heavy chain variable regionof SEQ ID NO: 21; a light chain variable region of SEQ ID NO: 9 and aheavy chain variable region of SEQ ID NO: 22; a light chain variableregion of SEQ ID NO: 10 and a heavy chain variable region of SEQ ID NO:23; a light chain variable region of SEQ ID NO: 11 and a heavy chainvariable region of SEQ ID NO: 24; a light chain variable region of SEQID NO: 12 and a heavy chain variable region of SEQ ID NO: 25; or a lightchain variable region of SEQ ID NO: 13 and a heavy chain variable regionof SEQ ID NO: 26. In other embodiments, the humanized immunoglobulinbinds to an epitope on human CD52 which overlaps with the epitope towhich such a mouse monoclonal antibody binds.

In other embodiments, the invention provides a humanized immunoglobulinwhich binds to an epitope on human CD52 (e.g., SEQ ID NO: 104)comprising at least residue 1 of the mature human CD52 sequence (whereresidue 1 is the N-terminus of the mature human CD52 sequence, i.e., theN-terminal glycine [G] residue; see FIG. 4). The humanizedimmunoglobulin may bind to an epitope comprising at least residues 1, 3,4 and 5 of the mature human CD52 sequence (these residues being aglycine [G], an asparagine [N], an aspartate [D], and a threonine [T],respectively). The humanized immunoglobulin may bind to an epitopecomprising at least residues 1, 2, 3, 4 and 5 of the mature human CD52sequence (these residues being a glycine [G], a glutamine [Q], anasparagine [N], an aspartate [D], and a threonine [T], respectively). Inother embodiments, the invention provides a humanized immunoglobulinwhich binds to an epitope on human CD52 comprising at least residues 7,8 and 9 of the mature human CD52 sequence (these residues being aglutamine [Q], a threonine [T], and a serine [S], respectively). In someembodiments, the epitope comprises at least residues 7 (Q), 8 (T) and 11(P) of the mature human CD52 sequence. In some embodiments, the epitopecomprises at least residues 4 (D) and 11 (P) of the mature human CD52sequence.

In some embodiments, the invention provides a humanized immunoglobulin,which binds to human CD52, and which comprises a light chain comprisingone or more CDRs selected from the group consisting of SEQ ID NO: 115,SEQ ID NO: 118, and SEQ ID NO: 121 (e.g., all three of said CDRs), or aheavy chain comprising one or more CDRs selected from the groupconsisting of SEQ ID NO: 124, SEQ ID NO: 127, and SEQ ID NO: 130 (e.g.,all three of said CDRs), or both such light chain and such heavy chain.In other embodiments, the invention provides a humanized immunoglobulin,which binds to human CD52, and which comprises a light chain comprisingone or more CDRs selected from the group consisting of SEQ ID NO: 116,SEQ ID NO: 119, and SEQ ID NO: 122 (e.g., all three of said CDRs), or aheavy chain comprising one or more CDRs selected from the groupconsisting of SEQ ID NO: 125, SEQ ID NO: 128, and SEQ ID NO: 131 (e.g.,all three of said CDRs), or both such light chain and heavy chain. Instill further embodiments, the invention provides a humanizedimmunoglobulin, which binds to human CD52, and which comprises a lightchain comprising one or more CDRs selected from the group consisting ofSEQ ID NO: 117, SEQ ID NO: 120, and SEQ ID NO: 123 (e.g., all three ofsaid CDRs), or a heavy chain comprising one or more CDRs selected fromthe group consisting of SEQ ID NO: 126, SEQ ID NO: 129, and SEQ ID NO:132 (e.g., all three of said CDRs), or both such light chain and suchheavy chains.

In certain embodiments, the humanized immunoglobulin comprises a lightchain comprising the CDRs of SEQ ID NO: 115, SEQ ID NO: 118 and SEQ IDNO: 121 and a heavy chain comprising the CDRs of SEQ ID NO: 124, SEQ IDNO: 127 and SEQ ID NO: 130. In other embodiments, the humanizedimmunoglobulin comprises a light chain comprising the CDRs of SEQ ID NO:116, SEQ ID NO: 119 and SEQ ID NO: 122 and a heavy chain comprising theCDRs of SEQ ID NO: 125, SEQ ID NO: 128 and SEQ ID NO: 131. In otherembodiments, the humanized immunoglobulin comprises a light chaincomprising the CDRs of SEQ ID NO: 117, SEQ ID NO: 120 and SEQ ID NO: 123and a heavy chain comprising the CDRs of SEQ ID NO: 126, SEQ ID NO: 129and SEQ ID NO: 132.

The humanized immunoglobulins of the present invention are differentfrom the humanized immunoglobulin Campath®.

The amino acid sequences of the above-mentioned SEQ ID NOs: 115-132 areprovided below, and are based on the amino acid sequences that arereported in Tables 1-6 as provided elsewhere herein. In these amino acidsequences, “X” stands for any amino acid, and the symbol “/” indicatesthat either (or any) of the amino acids depicted adjacent that symbolmay be present at the indicated position (e.g., K/R indicates that alysine or arginine residue is present at the indicated position andF/L/V indicates that a phenylalanine, leucine or valine residue ispresent at the indicated position).

Light Chain CDR-1 Sequences (SEQ ID NO: 115)K/RSSQSLL/V/IXS/TN/DGXS/TYLX (SEQ ID NO: 116) K/RSSQSLL/V/IHS/TNGXS/TYLH(SEQ ID NO: 117) RSSQSLVHTNGNS/TYLH Light Chain CDR-2 Sequences(SEQ ID NO: 118) XVSXXXS (SEQ ID NO: 119) XVSXRXS (SEQ ID NO: 120)MVSXRFS Light Chain CDR-3 Sequences (SEQ ID NO: 121) XQXXH/R/KF/L/V/IXX(SEQ ID NO: 122) SQSXH/R/KF/L/V/IPX (SEQ ID NO: 123) SQSXHVPF/PHeavy Chain CDR-1 Sequences (SEQ ID NO: 124) GFXFXXYW/YMX(SEQ ID NO: 125) GFTFXXYW/YMX (SEQ ID NO: 126) GFTFTDYW/YMSHeavy Chain CDR-2 Sequences (SEQ ID NO: 127) XIRXKXBXYXTXYXXSVKG(SEQ ID NO: 128) XIRXKXNXYTTEYXXSVKG (SEQ ID NO: 129)FIRNKANGYTTEYXXSVKG Heavy Chain CDR-3 Sequences (SEQ ID NO: 130)TXXXY/F/W (SEQ ID NO: 131) TRYXY/F/WFDY (SEQ ID NO: 132) TRYIF/WFDY

The invention also relates to a humanized light chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 115, SEQ IDNO: 118, and SEQ ID NO: 121 (e.g., all three of said CDRs); a humanizedlight chain comprising one or more CDRs selected from the groupconsisting of SEQ ID NO: 116, SEQ ID NO: 119, and SEQ ID NO: 122 (e.g.,all three of said CDRs); or a humanized light chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 117, SEQ IDNO: 120, and SEQ ID NO: 123 (e.g., all three of said CDRs).

The invention also relates to a humanized heavy chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 124, SEQ IDNO: 127, and SEQ ID NO: 130 (e.g., all three of said CDRs); a humanizedheavy chain comprising one or more CDRs selected from the groupconsisting of SEQ ID NO: 125, SEQ ID NO: 128, and SEQ ID NO: 131 (e.g.,all three of said CDRs); or a humanized heavy chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 126, SEQ IDNO: 129, and SEQ ID NO: 132 (e.g., all three of said CDRs).

The humanized light chains and humanized heavy chains of the presentinvention are different from the humanized light chain and the humanizedheavy chains of the humanized immunoglobulin Campath®.

In some embodiments of the present invention, the humanizedimmunoglobulins of the invention (irrespective of the manner in whichthey might otherwise be defined, e.g., regardless of whether they mightalso be defined in terms of the sequence of one or more of their CDRsand/or by their cross-reactivity with a mouse monoclonal antibody oranother humanized immunoglobulin): (1) exhibit binding to glycosylatedand de-glycosylated CD52 with no apparent preference; (2) exhibitbinding specific for glycosylated CD52; (3) exhibit binding specific forde-glycosylated CD52; or (4) exhibit binding preferential forde-glycosylated over glycosylated CD52. In certain embodiments, thehumanized immunoglobulins of the invention have a greater bindingaffinity for glycosylated human CD52 than for non-glycosylated orde-glycosylated human CD52. Indeed, in certain embodiments of thepresent invention, the humanized immunoglobulins of the presentinvention exhibit binding that is specific for glycosylated human CD52.Binding affinity for non-glycosylated or de-glycosylated human CD52 maybe determined with the use of mature human CD52 that has beende-glycosylated using a glycosidase, e.g., using the endoglycosidasePNGase-F. In certain embodiments of the present invention, the humanizedimmunoglobulins of the invention bind to an epitope on mature human CD52which comprises its N-linked carbohydrate moiety. This carbohydratemoiety is a sialylted, polylactosamine-containing core-fucosylatedtetraantennary N-linked oligosaccharide (Treumann, A. et al., (1995) J.Biol. Chem. 270:6088-6099). This epitope may also comprise at leastresidue 1 of the mature human CD52 sequence, at least residue 3 of themature human CD52 sequence, at least residues 1, 3, 4 and 5 of themature human CD52 sequence, or at least residues 1, 2, 3, 4 and 5 of themature human CD52 sequence. In some embodiments, the mouse or chimericantibodies of the present invention may have any of these bindingfeatures.

Isolated nucleic acid molecules that encode a humanized immunoglobulin,humanized light chain or humanized heavy chain of the invention, asdefined elsewhere herein, are also provided. In some embodiments, theinvention is an (one or more) isolated nucleic acid molecule encoding ahumanized heavy chain and a humanized light chain which associatetogether to form a humanized immunoglobulin that has binding specificityfor human CD52, wherein the humanized light chain comprises one or moreCDRs (e.g., all three CDRs) of SEQ ID NO: 3 and a heavy chain comprisingone or more CDRs (e.g., all three CDRs) of SEQ ID NO: 16; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 4 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 17; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 5 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 18; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 6 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 19; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 7 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 20; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 8 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 21; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 9 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 22; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 10 anda heavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQID NO: 23; a light chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 11 and a heavy chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 24; a light chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 12 and a heavy chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 25; alight chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 12 and a heavy chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 137; or a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 13 and a heavy chain sequencecomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 26.

In some embodiments, the invention is one or more isolated nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulinbinds to the same epitope on human CD52 as a mouse monoclonal antibodycomprising a light chain variable region of SEQ ID NO: 3 and a heavychain variable region of SEQ ID NO: 16; a light chain variable region ofSEQ ID NO: 4 and a heavy chain variable region of SEQ ID NO: 17; a lightchain variable region of SEQ ID NO: 5 and a heavy chain variable regionof SEQ ID NO: 18; a light chain variable region of SEQ ID NO: 6 and aheavy chain variable region of SEQ ID NO: 19; a light chain variableregion of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO:20; a light chain variable region of SEQ ID NO: 8 and a heavy chainvariable region of SEQ ID NO: 21; a light chain variable region of SEQID NO: 9 and a heavy chain variable region of SEQ ID NO: 22; a lightchain variable region of SEQ ID NO: 10 and a heavy chain variable regionof SEQ ID NO: 23; a light chain variable region of SEQ ID NO: 11 and aheavy chain variable region of SEQ ID NO: 24; a light chain variableregion of SEQ ID NO: 12 and a heavy chain variable region of SEQ ID NO:25; or a light chain variable region of SEQ ID NO: 13 and a heavy chainvariable region of SEQ ID NO: 26. In other embodiments, the invention isone or more isolated nucleic acid molecules encoding a humanized heavychain and a humanized light chain which associate together to form ahumanized immunoglobulin that has binding specificity for human CD52,wherein the humanized immunoglobulin binds to an epitope on human CD52which overlaps with the epitope to which such a mouse monoclonalantibody binds.

In other embodiments, the invention is one or more isolated nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulinbinds to an epitope comprising at least residue 1 of mature human CD52;the humanized immunoglobulin binds to an epitope comprising at leastresidues 1, 3, 4 and 5 of mature human CD52; the humanizedimmunoglobulin binds to an epitope comprising at least residues 1, 2, 3,4 and 5 of mature human CD52; or the humanized immunoglobulin binds toan epitope comprising at least residues 7, 8 and 9 of mature human CD52.In some embodiments, the epitope comprises at least residues 7, 8 and 11of the mature human CD52 sequence. In some embodiments, the epitopecomprises at least residues 4 and 11 of the mature human CD52 sequence.

In other embodiments, the invention is one or more isolated nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulincomprises a light chain comprising one or more CDRs selected from thegroup consisting of SEQ ID NO: 115, SEQ ID NO: 118, and SEQ ID NO: 121(e.g., all three of said CDRs), and/or a heavy chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 124, SEQ IDNO: 127, and SEQ ID NO: 130 (e.g., all three of said CDRs); a lightchain comprising one or more CDRs selected from the group consisting ofSEQ ID NO: 116, SEQ ID NO: 119, and SEQ ID NO: 122 (e.g., all three ofsaid CDRs), and/or a heavy chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 125, SEQ ID NO: 128, and SEQ IDNO: 131 (e.g., all three of said CDRs); or a light chain comprising oneor more CDRs selected from the group consisting of SEQ ID NO: 117, SEQID NO: 120, and SEQ ID NO: 123 (e.g., all three of said CDRs), and/or aheavy chain comprising one or more CDRs selected from the groupconsisting of SEQ ID NO: 126, SEQ ID NO: 129, and SEQ ID NO: 132 (e.g.,all three of said CDRs).

In certain embodiments, the invention is one or more isolated nucleicacid molecules encoding a humanized heavy chain and a humanized lightchain which associate together to form a humanized immunoglobulin thathas binding specificity for human CD52, wherein the humanizedimmunoglobulin comprises a light chain comprising the CDRs of SEQ ID NO:115, SEQ ID NO: 118 and SEQ ID NO: 121 and a heavy chain comprising theCDRs of SEQ ID NO: 124, SEQ ID NO: 127 and SEQ ID NO: 130; a light chaincomprising the CDRs of SEQ ID NO: 116, SEQ ID NO: 119 and SEQ ID NO: 122and a heavy chain comprising the CDRs of SEQ ID NO: 125, SEQ ID NO: 128and SEQ ID NO: 131; or a light chain comprising the CDRs of SEQ ID NO:117, SEQ ID NO: 120 and SEQ ID NO: 123 and a heavy chain comprising theCDRs of SEQ ID NO: 126, SEQ ID NO: 129 and SEQ ID NO: 132.

The one or more nucleic acids of the invention do not encode thehumanized immunoglobulin Campath®.

In other embodiments, the invention is one or more isolated nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulinhas a greater binding affinity for glycosylated human CD52 than fornon-glycosylated or de-glycosylated human CD52, e.g., exhibits bindingthat is specific for glycosylated human CD52. The humanizedimmunoglobulin may bind to an epitope on mature human CD52 whichcomprises its N-linked carbohydrate moiety. This epitope may alsocomprise at least residue 1 of the mature human CD52 sequence, at leastresidue 3 of the mature human CD52 sequence, at least residues 1, 3, 4and 5 of the mature human CD52 sequence, or at least residues 1, 2, 3, 4and 5 of the mature human CD52 sequence.

In other embodiments, the invention is an isolated nucleic acid moleculeencoding a humanized light chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12 or SEQ ID NO: 13, wherein the humanized light chain is notthe humanized light chain of Campath®.

In other embodiments, the invention is an isolated nucleic acid moleculeencoding a humanized heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 137, wherein thehumanized heavy chain is not the humanized heavy chain of Campath®.

In other embodiments, the invention is an isolated nucleic acid moleculeencoding a humanized light chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ IDNO: 48, or a combination thereof, wherein the humanized light chain isnot the humanized light chain of Campath®.

In other embodiments, the invention is an isolated nucleic acid moleculeencoding a humanized heavy chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ IDNO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65,SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and SEQID NO: 294, or a combination thereof, wherein the humanized heavy chainis not the humanized heavy chain of Campath®.

In other embodiments, the invention is an isolated nucleic acid moleculeencoding a humanized light chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 115, SEQ ID NO: 118, and SEQ IDNO: 121 (e.g., all three of said CDRs); a humanized light chaincomprising one or more CDRs selected from the group consisting of SEQ IDNO: 116, SEQ ID NO: 119, and SEQ ID NO: 122 (e.g., all three of saidCDRs); or a humanized light chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 117, SEQ ID NO: 120, and SEQ IDNO: 123 (e.g., all three of said CDRs).

In other embodiments, the invention is an isolated nucleic acid moleculeencoding a humanized heavy chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 124, SEQ ID NO: 127, and SEQ IDNO: 130 (e.g., all three of said CDRs); a humanized heavy chaincomprising one or more CDRs selected from the group consisting of SEQ IDNO: 125, SEQ ID NO: 128, and SEQ ID NO: 131 (e.g., all three of saidCDRs); or a humanized heavy chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 126, SEQ ID NO: 129, and SEQ IDNO: 132 (e.g., all three of said CDRs).

The invention also relates to recombinant vectors (e.g., expressionvectors, including mammalian cell expression vectors) that comprise anucleic acid encoding a humanized immunoglobulin (e.g., a humanizedlight chain and a humanized heavy chain), a humanized light chain, or ahumanized heavy chain of the invention. In some embodiments, theinvention is a recombinant vector comprising a nucleic acid encoding ahumanized immunoglobulin that comprises a light chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 3 and a heavy chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 16; alight chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 4 and a heavy chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 17; a light chain comprising one or more CDRs (e.g.,all three CDRs) of SEQ ID NO: 5 and a heavy chain comprising one or moreCDRs (e.g., all three CDRs) of SEQ ID NO: 18; a light chain comprisingone or more CDRs (e.g., all three CDRs) of SEQ ID NO: 6 and a heavychain comprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO:19; a light chain comprising one or more CDRs (e.g., all three CDRs) ofSEQ ID NO: 7 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 20; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 8 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 21; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 9 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 22; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 10 and a heavy chain comprising one or more CDRs (e.g.,all three CDRs) of SEQ ID NO: 23; a light chain comprising one or moreCDRs (e.g., all three CDRs) of SEQ ID NO: 11 and a heavy chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 24; alight chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 12 and a heavy chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 25; a light chain comprising one or more CDRs (e.g.,all three CDRs) of SEQ ID NO: 12 and a heavy chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 137; or a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 13 anda heavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQID NO: 26.

In other embodiments, the recombinant vector comprises a nucleic acidencoding a humanized light chain, wherein the humanized light chaincomprises one or more CDRs selected from the group consisting of SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ IDNO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, or a combination thereof,wherein the humanized light chain is not the humanized light chain ofCampath®.

In other embodiments, the recombinant vector comprises a nucleic acidencoding a humanized heavy chain, wherein the humanized heavy chaincomprises one or more CDRs selected from the group consisting of SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ IDNO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 294, or a combination thereof,wherein the humanized light chain is not the humanized light chain ofCampath®.

In some embodiments, the invention provides a recombinant vectorcomprising a nucleic acid molecule, or a pair of recombinant vectorscomprising nucleic acid molecules, encoding a humanized heavy chain anda humanized light chain which associate together to form a humanizedimmunoglobulin that has binding specificity for human CD52, wherein thehumanized immunoglobulin binds to the same epitope on human CD52 as amouse monoclonal antibody comprising a light chain variable region ofSEQ ID NO: 3 and a heavy chain variable region of SEQ ID NO: 16; a lightchain variable region of SEQ ID NO: 4 and a heavy chain variable regionof SEQ ID NO: 17; a light chain variable region of SEQ ID NO: 5 and aheavy chain variable region of SEQ ID NO: 18; a light chain variableregion of SEQ ID NO: 6 and a heavy chain variable region of SEQ ID NO:19; a light chain variable region of SEQ ID NO: 7 and a heavy chainvariable region of SEQ ID NO: 20; a light chain variable region of SEQID NO: 8 and a heavy chain variable region of SEQ ID NO: 21; a lightchain variable region of SEQ ID NO: 9 and a heavy chain variable regionof SEQ ID NO: 22; a light chain variable region of SEQ ID NO: 10 and aheavy chain variable region of SEQ ID NO: 23; a light chain variableregion of SEQ ID NO: 11 and a heavy chain variable region of SEQ ID NO:24; a light chain variable region of SEQ ID NO: 12 and a heavy chainvariable region of SEQ ID NO: 25; or a light chain variable region ofSEQ ID NO: 13 and a heavy chain variable region of SEQ ID NO: 26. Inother embodiments, the invention provides a recombinant vectorcomprising a nucleic acid molecule, or a pair of recombinant vectorscomprising nucleic acid molecules, encoding a humanized heavy chain anda humanized light chain which associate together to form a humanizedimmunoglobulin that has binding specificity for human CD52, wherein thehumanized immunoglobulin binds to an epitope on human CD52 whichoverlaps with the epitope to which such a mouse monoclonal antibodybinds.

In other embodiments, the recombinant vector comprises a nucleic acidmolecule, or a pair of recombinant vectors comprise nucleic acidmolecules, encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulinbinds to an epitope comprising at least residue 1 of mature human CD52;binds to an epitope comprising at least residues 1, 3, 4 and 5 of maturehuman CD52; binds to an epitope comprising at least residues 1, 2, 3, 4and 5 of mature human CD52; or binds to an epitope comprising at leastresidues 7, 8 and 9 of mature human CD52. In some embodiments, theepitope comprises at least residues 7, 8 and 11 of the mature human CD52sequence. In some embodiments, the epitope comprises at least residues 4and 11 of the mature human CD52 sequence.

In some embodiments, the recombinant vector comprises a nucleic acidmolecule, or a pair of recombinant vectors comprise nucleic acidmolecules, encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulincomprises a light chain comprising one or more CDRs selected from thegroup consisting of SEQ ID NO: 115, SEQ ID NO: 118, and SEQ ID NO: 121(e.g., all three of said CDRs), and/or a heavy chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 124, SEQ IDNO: 127, and SEQ ID NO: 130 (e.g., all three of said CDRs); a lightchain comprising one or more CDRs selected from the group consisting ofSEQ ID NO: 116, SEQ ID NO: 119, and SEQ ID NO: 122 (e.g., all three ofsaid CDRs), and/or a heavy chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 125, SEQ ID NO: 128, and SEQ IDNO: 131 (e.g., all three of said CDRs); or a light chain comprising oneor more CDRs selected from the group consisting of SEQ ID NO: 117, SEQID NO: 120, and SEQ ID NO: 123 (e.g., all three of said CDRs), and/or aheavy chain comprising one or more CDRs selected from the groupconsisting of SEQ ID NO: 126, SEQ ID NO: 129, and SEQ ID NO: 132 (e.g.,all three of said CDRs).

In certain embodiments, the recombinant vector comprises a nucleic acidmolecule, or a pair of recombinant vectors comprise nucleic acidmolecules, encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulincomprises a light chain comprising the CDRs of SEQ ID NO: 115, SEQ IDNO: 118 and SEQ ID NO: 121 and a heavy chain comprising the CDRs of SEQID NO: 124, SEQ ID NO: 127 and SEQ ID NO: 130; a light chain comprisingthe CDRs of SEQ ID NO: 116, SEQ ID NO: 119 and SEQ ID NO: 122 and aheavy chain comprising the CDRs of SEQ ID NO: 125, SEQ ID NO: 128 andSEQ ID NO: 131; or a light chain comprising the CDRs of SEQ ID NO: 117,SEQ ID NO: 120 and SEQ ID NO: 123 and a heavy chain comprising the CDRsof SEQ ID NO: 126, SEQ ID NO: 129 and SEQ ID NO: 132.

The one or more nucleic acids in the recombinant vector or vectors ofthe present invention do not encode the humanized immunoglobulinCampath®.

In other embodiments, the recombinant vector comprises a nucleic acidmolecule, or a pair of recombinant vectors comprise nucleic acidmolecules, encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulinhas a greater binding affinity for glycosylated human CD52 than fornon-glycosylated or de-glycosylated human CD52, e.g., exhibits bindingthat is specific for glycosylated human CD52. The humanizedimmunoglobulin may bind to an epitope on mature human CD52 whichcomprises its N-linked carbohydrate moiety. This epitope may alsocomprise at least residue 1 of the mature human CD52 sequence, at leastresidue 3 of the mature human CD52 sequence, at least residues 1, 3, 4and 5 of the mature human CD52 sequence, or at least residues 1, 2, 3, 4and 5 of the mature human CD52 sequence.

In other embodiments, the recombinant vector comprises a nucleic acidmolecule encoding a humanized light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, wherein the humanized lightchain is not the humanized light chain of Campath®.

In other embodiments, the recombinant vector comprises a nucleic acidmolecule encoding a humanized heavy chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 137,wherein the humanized heavy chain is not the humanized heavy chain ofCampath®.

In other embodiments, the recombinant vector comprises a nucleic acidmolecule encoding a humanized light chain comprising one or more CDRsselected from the group consisting of SEQ ID NO: 115, SEQ ID NO: 118,and SEQ ID NO: 121 (e.g., all three of said CDRs); a humanized lightchain comprising one or more CDRs selected from the group consisting ofSEQ ID NO: 116, SEQ ID NO: 119, and SEQ ID NO: 122 (e.g., all three ofsaid CDRs); or a humanized light chain comprising one or more CDRsselected from the group consisting of SEQ ID NO: 117, SEQ ID NO: 120,and SEQ ID NO: 123 (e.g., all three of said CDRs), wherein the humanizedlight chain is not the humanized light chain of Campath®.

In other embodiments, the recombinant vector comprises a nucleic acidmolecule encoding a humanized heavy chain comprising one or more CDRsselected from the group consisting of SEQ ID NO: 124, SEQ ID NO: 127,and SEQ ID NO: 130 (e.g., all three of said CDRs); a humanized heavychain comprising one or more CDRs selected from the group consisting ofSEQ ID NO: 125, SEQ ID NO: 128, and SEQ ID NO: 131 (e.g., all three ofsaid CDRs); or a humanized heavy chain comprising one or more CDRsselected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 129,and SEQ ID NO: 132 (e.g., all three of said CDRs), wherein the humanizedheavy chain is not the humanized heavy chain of Campath®.

In particular embodiments, the recombinant vector of the invention is anexpression vector, such as a mammalian cell expression vector. Incertain embodiments, the vector is a plasmid or a viral vector (e.g., anadenoviral or AAV vector).

The invention also relates to a host cell that comprises a (one or more)nucleic acid (e.g., recombinant) encoding a humanized immunoglobulin(humanized light chain and humanized heavy chain), a humanized lightchain or a humanized heavy chain of the invention. In some embodiments,the host cell comprises a recombinant vector (e.g., expression vector,including mammalian cell expression vectors) of the invention.

In a particular embodiment, the host cell comprises a nucleic acid (oneor more nucleic acids) encoding a humanized light chain and a humanizedheavy chain, wherein the humanized light chain and the humanized heavychain associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52 and wherein the humanizedimmunoglobulin comprises a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 3 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 16; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 4 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 17; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 5 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 18; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 6 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 19; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 7 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 20; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 8 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 21; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 9 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 22; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 10 anda heavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQID NO: 23; a light chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 11 and a heavy chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 24; a light chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 12 and a heavy chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 25; alight chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 12 and a heavy chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 137; or a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 13 and a heavy chain sequencecomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 26.

In some embodiments, the host cell comprises one or more nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulinbinds to the same epitope on human CD52 as a mouse monoclonal antibodycomprising a light chain variable region of SEQ ID NO: 3 and a heavychain variable region of SEQ ID NO: 16; a light chain variable region ofSEQ ID NO: 4 and a heavy chain variable region of SEQ ID NO: 17; a lightchain variable region of SEQ ID NO: 5 and a heavy chain variable regionof SEQ ID NO: 18; a light chain variable region of SEQ ID NO: 6 and aheavy chain variable region of SEQ ID NO: 19; a light chain variableregion of SEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO:20; a light chain variable region of SEQ ID NO: 8 and a heavy chainvariable region of SEQ ID NO: 21; a light chain variable region of SEQID NO: 9 and a heavy chain variable region of SEQ ID NO: 22; a lightchain variable region of SEQ ID NO: 10 and a heavy chain variable regionof SEQ ID NO: 23; a light chain variable region of SEQ ID NO: 11 and aheavy chain variable region of SEQ ID NO: 24; a light chain variableregion of SEQ ID NO: 12 and a heavy chain variable region of SEQ ID NO:25; or a light chain variable region of SEQ ID NO: 13 and a heavy chainvariable region of SEQ ID NO: 26. In other embodiments, the host cellcomprises one or more nucleic acid molecules encoding a humanized heavychain and a humanized light chain which associate together to form ahumanized immunoglobulin that has binding specificity for human CD52,wherein the humanized immunoglobulin binds to an epitope on human CD52which overlaps with the epitope to which such a mouse monoclonalantibody binds.

In other embodiments, the host cell comprises one or more nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulinbinds to an epitope comprising at least residue 1 of mature human CD52;binds to an epitope comprising at least residues 1, 3, 4 and 5 of maturehuman CD52; binds to an epitope comprising at least residues 1, 2, 3, 4and 5 of mature human CD52; or binds to an epitope comprising at leastresidues 7, 8 and 9 of mature human CD52. In some embodiments, theepitope comprises at least residues 7, 8 and 11 of the mature human CD52sequence. In some embodiments, the epitope comprises at least residues 4and 11 of the mature human CD52 sequence.

In some embodiments, the host cell comprises one or more nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulincomprises a light chain comprising one or more CDRs selected from thegroup consisting of SEQ ID NO: 115, SEQ ID NO: 118, and SEQ ID NO: 121(e.g., all three of said CDRs), and/or a heavy chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 124, SEQ IDNO: 127, and SEQ ID NO: 130 (e.g., all three of said CDRs); a lightchain comprising one or more CDRs selected from the group consisting ofSEQ ID NO: 116, SEQ ID NO: 119, and SEQ ID NO: 122 (e.g., all three ofsaid CDRs), and/or a heavy chain comprising one or more CDRs selectedfrom the group consisting of SEQ ID NO: 125, SEQ ID NO: 128, and SEQ IDNO: 131 (e.g., all three of said CDRs); or a light chain comprising oneor more CDRs selected from the group consisting of SEQ ID NO: 117, SEQID NO: 120, and SEQ ID NO: 123 (e.g., all three of said CDRs), and/or aheavy chain comprising one or more CDRs selected from the groupconsisting of SEQ ID NO: 126, SEQ ID NO: 129, and SEQ ID NO: 132 (e.g.,all three of said CDRs).

In some embodiments, the host cell comprises one or more nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulincomprises a light chain comprising the CDRs of SEQ ID NO: 115, SEQ IDNO: 118 and SEQ ID NO: 121 and a heavy chain comprising the CDRs of SEQID NO: 124, SEQ ID NO: 127 and SEQ ID NO: 130; a light chain comprisingthe CDRs of SEQ ID NO: 116, SEQ ID NO: 119 and SEQ ID NO: 122 and aheavy chain comprising the CDRs of SEQ ID NO: 125, SEQ ID NO: 128 andSEQ ID NO: 131; or a light chain comprising the CDRs of SEQ ID NO: 117,SEQ ID NO: 120 and SEQ ID NO: 123 and a heavy chain comprising the CDRsof SEQ ID NO: 126, SEQ ID NO: 129 and SEQ ID NO: 132.

In other embodiments, the host cell comprises one or more nucleic acidmolecules encoding a humanized heavy chain and a humanized light chainwhich associate together to form a humanized immunoglobulin that hasbinding specificity for human CD52, wherein the humanized immunoglobulinhas a greater binding affinity for glycosylated human CD52 than fornon-glycosylated or de-glycosylated human CD52, e.g., exhibits bindingthat is specific for glycosylated human CD52. The humanizedimmunoglobulin may bind to an epitope on mature human CD52 whichcomprises its N-linked carbohydrate moiety. This epitope may alsocomprise at least residue 1 of the mature human CD52 sequence, at leastresidue 3 of the mature human CD52 sequence, at least residues 1, 3, 4and 5 of the mature human CD52 sequence, or at least residues 1, 2, 3, 4and 5 of the mature human CD52 sequence.

In some embodiments, the host cell comprises a nucleic acid moleculeencoding a humanized light chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12 or SEQ ID NO: 13. The humanized light chain is not thehumanized light chain of Campath®.

In other embodiments, the host cell comprises a nucleic acid moleculeencoding a humanized heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 137. The humanizedheavy chain is not the humanized heavy chain of Campath®.

In some embodiments, the host cell comprises a nucleic acid encoding ahumanized light chain, wherein the humanized light chain comprises oneor more CDRs selected from the group consisting of SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ IDNO: 47, and SEQ ID NO: 48 or a combination thereof, wherein thehumanized light chain is not the humanized light chain of Campath®.

In other embodiments, the host cell comprises a nucleic acid encoding ahumanized heavy chain, wherein the humanized heavy chain comprises oneor more CDRs selected from the group consisting of SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 74, and SEQ ID NO: 294, or a combination thereof, wherein thehumanized heavy chain is not the humanized heavy chain of Campath®.

The invention also relates to a method of preparing a humanizedimmunoglobulin that has binding specificity for human CD52 comprisingmaintaining a host cell of the invention (e.g., a host cell thatcontains one or more recombinant nucleic acids that encode a humanizedimmunoglobulin of the invention (e.g., a humanized light chain and ahumanized heavy chain of the invention)) under conditions appropriatefor expression of a humanized immunoglobulin, whereby humanizedimmunoglobulin chains are expressed and a humanized immunoglobulin isproduced. In some embodiments, the method further comprises purifying orisolating the humanized immunoglobulin. In some embodiments, the methodfurther comprises combining the purified or isolated humanizedimmunoglobulin with a physiologically acceptable vehicle or carrier toproduce a pharmaceutical composition.

The invention also relates to a method of preparing a humanized lightchain that has binding specificity for human CD52 comprising maintaininga host cell of the invention (e.g., a host cell that contains one ormore recombinant nucleic acids that encode a humanized light chain ofthe invention) under conditions appropriate for expression of ahumanized light chain, whereby a humanized light chain is expressed anda humanized light chain is produced. In some embodiments, the methodfurther comprises purifying or isolating the humanized light chain.

The invention also relates to a method of preparing a humanized heavychain that has binding specificity for human CD52 comprising maintaininga host cell of the invention (e.g., a host cell that contains one ormore recombinant nucleic acids that encode a humanized heavy chain ofthe invention) under conditions appropriate for expression of ahumanized heavy chain, whereby a humanized heavy chain is expressed anda humanized heavy chain is produced. In some embodiments, the methodfurther comprises purifying or isolating the humanized heavy chain.

The invention further relates to a pharmaceutical composition comprisinga humanized immunoglobulin of the invention (e.g., comprising ahumanized light chain of the invention and/or a humanized heavy chain ofthe invention) and a physiologically acceptable vehicle or carrier. Insome embodiments, the pharmaceutical composition comprises a unit dosecomposition.

The invention also relates to a method of producing a hybridoma thatsecretes a monoclonal antibody that has binding specificity for humanCD52 comprising administering lymphocytes of a mouse transgenic forhuman CD52 to a non-transgenic mouse of the same, or of a similar,strain (e.g., CD1) as the human CD52 transgenic mouse, thereby producingan immunized, non-transgenic mouse. Splenocytes of the immunized,non-transgenic mouse are fused with immortalized cells, therebyproducing a hybridoma. The hybridoma is maintained under conditions inwhich it will secrete a monoclonal antibody having binding specificityfor human CD52. In some embodiments, FACS analysis is used to detect ahybridoma that secretes a monoclonal antibody that has bindingspecificity for human CD52. In other embodiments, the strain of thetransgenic mouse and the strain of the non-transgenic mouse areidentical. In certain embodiments, the CD52 is wildtype human CD52. Insome embodiments, the CD52 transgenic mouse and the non-transgenic mouseare CD1 mice. In some embodiments, the lymphocytes used for immunizationare obtained from the spleen of the human CD52 transgenic mouse. In someembodiments, the immortalized cells are selected from the groupconsisting of SP2/0 Ag14 cells and NS1 myeloma cells. The invention alsorelates to a hybridoma produced by the methods of the invention.Optionally, the monoclonal antibody secreted by the hybridoma iscollected and can be further purified (e.g., substantially purified,isolated). In other embodiments, the method further comprisesdetermining the nucleotide sequence of the monoclonal antibody secretedby the hybridoma.

The invention also relates to a method for treating an autoimmunedisease (e.g., multiple sclerosis (MS), rheumatoid arthritis (RA) (Seee.g., Nature Reviews Drug Discovery 6: 75-92 (2007)), vasculitis (Seee.g., Rheumatology 39:229-237 (2000)), Behcet's disease (BD) (See e.g.,Rheumatology 42:1539-1544 (2003)), lupus and celiac disease (Vivas, S.,et al., N Engl. J. Med., 354(23):2514-2515 (2006)), vasculitis,psoriasis, myositis, scleroderma, aplastic anemia, and colitis) in apatient in need thereof, comprising administering to the patient aneffective amount of a humanized immunoglobulin of the invention.

In another aspect, an effective amount of a humanized immunoglobulin ofthe invention can be administered in conjunction with one or moreimmunosuppressive agents to prepare a patient in need thereof for asolid organ transplant (Agarwal et al., Transplant Immunol., 20:6-11(2008)) or a CD34+ stem cell transplant (Burt et al., The Lancet,published online Jan. 30, 2009).

The invention also relates to a method for treating cancer in a patientin need thereof, comprising administering to the patient an effectiveamount of a humanized immunoglobulin of the invention.

The invention also relates to a method for treating multiple sclerosisin a patient in need thereof, comprising administering to the patient aneffective amount of a humanized immunoglobulin of the invention.

The invention also relates to a method for treating chronic lymphocyticleukemia in a patient in need thereof, comprising administering to thepatient an effective amount of a humanized immunoglobulin of theinvention.

The administration of a humanized immunoglobulin of the presentinvention may comprise the administration of the humanizedimmunoglobulin per se (e.g., in a pharmaceutical composition), theadministration of one or more recombinant vectors encoding the humanizedimmunoglobulin, or the administration of a host cell which comprises oneor more nucleic acids (e.g., one or more recombinant vectors) encodingthe humanized immunoglobulins and expresses the humanizedimmunoglobulin.

The invention also relates to a method of diagnosing a disease selectedfrom the group consisting of autoimmune diseases (e.g., multiplesclerosis, lupus, vasculitis), cancer (e.g., leukemias (e.g., chroniclymphocytic leukemia), and lymphomas (e.g., non-Hodgkin's lymphoma)),transplant (e.g., solid organ transplant (e.g., kidney transplant) andstem cell transplant), ‘comprising assaying a patient sample in vitrowith a humanized immunoglobulin of the invention.

The invention also relates to a humanized immunoglobulin of theinvention (e.g., comprising a humanized light chain of the inventionand/or humanized heavy chain of the invention), a recombinant vector ofthe invention, or a host cell of the invention, for use in medicine,such as for use in therapy and/or diagnosis of a disease such as for usein treating a disease or disorder described herein such as an autoimmunedisease (e.g., multiple sclerosis, rheumatoid arthritis, and lupus),cancer, a lymphocyte hyper-proliferative condition (e.g., T or B cellmalignancies including leukemia such as B-cell chronic lymphocyticleukemia and lymphomas such as non-Hodgkin's lymphoma). See, e.g.,Lundin, J., et al., Blood, 101:4267-4272 (2003); Rodig, S J., et al.,Clinical Cancer Research, 12(23):7174-7179 (2006). The invention alsorelates to the use of a humanized immunoglobulin, humanized light chainor humanized heavy chain of the invention, a recombinant vector of theinvention, or a host cell of the invention, for the manufacture of amedicament for treating a disease or disorder described herein (e.g.,autoimmune diseases (e.g., multiple sclerosis, lupus, vasculitis),cancer (e.g., leukemias (e.g., chronic lymphocytic leukemia), andlymphomas (e.g., non-Hodgkin's lymphoma)), and transplant (e.g., solidorgan transplant (e.g., kidney transplant) and stem cell transplant)’).

The invention further provides humanized anti-human CD52 antibodiescomprising human light chain framework regions that utilize a humanVk2-A18b gene in which residues 36 (Y) and 46 (L) (Kabat numbering) havebeen substituted. In some embodiments, residue 36 is V or L and residue46 is R. The invention also provides humanized anti-human CD52antibodies comprising human heavy chain framework regions that utilize ahuman VH 3-23 gene in which residue 47 (W) (Kabat numbering) has beensubstituted. In some embodiments, residues 47 (W) and 49 (S) (Kabatnumbering) both have been substituted. In some embodiments, residue 47is L and residue 49 is S. In other embodiments, residue 47 is L andresidue 49 is A.

In some embodiments, a humanized anti-human CD52 antibody of theinvention has an EC₅₀ value as determined in a cell-binding assay suchas the assay described in Example 29 that is two-fold lower than theEC₅₀ value for Campath-1H® antibody. In various embodiments, thehumanized anti-human CD52 antibody has an EC₅₀ value of 11 nM or less.

In some embodiments, a humanized anti-human CD52 antibody of theinvention binds CD52 on cells in the presence of anti-Campath-1H®antibodies from the serum of a human patient who has been treated withCampath-1H®. That is, the binding of a humanized anti-human CD52antibody of the invention to CD52 on cells is not reduced in thepresence of such anti-Campath-1H® antibodies compared to Campath-1H®binding to CD52 or is less reduced in the presence of suchanti-Campath-1H® antibodies compared to Campath-1H® binding to CD52.

The invention further provides humanized anti-human CD52 antibodies witha lymphocyte depletion profile in blood and/or spleen of a humanizedanti-human CD52 antibody provided herein.

In some embodiments, a humanized anti-human CD52 antibody of theinvention increases the circulating level of one or more of TNFalpha,IL-6 and MCP-1 in the serum of a subject.

In some embodiments, a humanized anti-human CD52 antibody of theinvention reduces lymphocyte levels in a subject for at least 30 days,at least 50 days, at least 60 days, at least 70 days, at least 80 daysor for more than 80 days.

In some embodiments, a humanized anti-human CD52 antibody of theinvention delays the onset of disease and/or decreases the severity ofdisease as measured by clinical score in a mouse EAE model.

In some embodiments, a humanized anti-human CD52 antibody of theinvention is less immunogenic than Campath-1H® in an immunogenicityassay such as the assay described in Example 69 or 70.

Mouse Monoclonal Immunoglobulins

The invention also relates to mouse monoclonal antibodies (mousemonoclonal immunoglobulins) that have binding specificity for humanCD52. In one embodiment, the invention relates to a mouse monoclonalantibody that has binding specificity for human CD52, comprising a lightchain comprising SEQ ID NO: 3 and a heavy chain comprising SEQ ID NO:16; a light chain comprising SEQ ID NO: 4 and a heavy chain comprisingSEQ ID NO: 17; a light chain comprising SEQ ID NO: 5 and a heavy chaincomprising SEQ ID NO: 18; a light chain comprising SEQ ID NO: 6 and aheavy chain comprising SEQ ID NO: 19; a light chain comprising SEQ IDNO: 7 and a heavy chain comprising SEQ ID NO: 20; a light chaincomprising SEQ ID NO: 8 and a heavy chain comprising SEQ ID NO: 21; alight chain comprising SEQ ID NO: 9 and a heavy chain comprising SEQ IDNO: 22; a light chain comprising SEQ ID NO: 10 and a heavy chaincomprising SEQ ID NO: 23; a light chain comprising SEQ ID NO: 11 and aheavy chain comprising SEQ ID NO: 24; a light chain comprising SEQ IDNO: 12 and a heavy chain comprising SEQ ID NO: 25; or a light chaincomprising SEQ ID NO: 13 and a heavy chain comprising SEQ ID NO: 26.

In one embodiment, the mouse monoclonal antibody that has bindingspecificity for human CD52 comprises a light chain variable regionselected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, or a heavychain variable region selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, andSEQ ID NO: 26, or both such light chain variable region and such heavychain variable region.

The invention also relates to a mouse immunoglobulin light chaincomprising the variable region of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

The invention also relates to a mouse immunoglobulin heavy chaincomprising the variable region of SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

Preferably, the mouse monoclonal antibodies of the present inventioncomprise both a mouse antibody light chain of the invention and a mouseantibody heavy chain of the invention. In some embodiments, theinvention provides a mouse monoclonal immunoglobulin which binds to thesame epitope on human CD52 as a mouse monoclonal antibody comprising alight chain variable region of SEQ ID NO: 3 and a heavy chain variableregion of SEQ ID NO: 16; a light chain variable region of SEQ ID NO: 4and a heavy chain variable region of SEQ ID NO: 17; a light chainvariable region of SEQ ID NO: 5 and a heavy chain variable region of SEQID NO: 18; a light chain variable region of SEQ ID NO: 6 and a heavychain variable region of SEQ ID NO: 19; a light chain variable region ofSEQ ID NO: 7 and a heavy chain variable region of SEQ ID NO: 20; a lightchain variable region of SEQ ID NO: 8 and a heavy chain variable regionof SEQ ID NO: 21; a light chain variable region of SEQ ID NO: 9 and aheavy chain variable region of SEQ ID NO: 22; a light chain variableregion of SEQ ID NO: 10 and a heavy chain variable region of SEQ ID NO:23; a light chain variable region of SEQ ID NO: 11 and a heavy chainvariable region of SEQ ID NO: 24; a light chain variable region of SEQID NO: 12 and a heavy chain variable region of SEQ ID NO: 25; or a lightchain variable region of SEQ ID NO: 13 and a heavy chain variable regionof SEQ ID NO: 26. In other embodiments, the invention provides a mousemonoclonal immunoglobulin which binds to an epitope on human CD52 whichoverlaps with the epitope to which such a mouse monoclonal antibodybinds.

In other embodiments, the invention provides a mouse monoclonalimmunoglobulin which binds to an epitope on human CD52 comprising atleast residue 1 of the mature human CD52 sequence. The mouse monoclonalimmunoglobulin may bind to an epitope comprising at least residues 1, 3,4 and 5 of the mature human CD52 sequence, may bind to an epitopecomprising at least residues 1, 2, 3, 4 and 5 of the mature human CD52sequence, or may bind to an epitope comprising at least residues 7, 8and 9 of the mature human CD52 sequence. In some embodiments, theepitope comprises at least residues 7, 8 and 11 of the mature human CD52sequence. In some embodiments, the epitope comprises at least residues 4and 11 of the mature human CD52 sequence.

The invention also relates to isolated nucleic acid molecules thatencode the mouse monoclonal immunoglobulins, mouse immunoglobulin lightchains or mouse immunoglobulin heavy chains of the invention. In someembodiments, the invention is an isolated nucleic acid molecule encodinga mouse immunoglobulin heavy chain and a mouse immunoglobulin lightchain which associate together to form a mouse monoclonal immunoglobulinthat has binding specificity for human CD52, wherein the mouseimmunoglobulin light chain comprises a variable region selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12 and SEQ ID NO: 13, or the mouse immunoglobulin heavychain comprises a variable region selected from the group consisting ofSEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25 and SEQ ID NO: 26, or both such light chain and such heavy chain.

In some embodiments, the isolated nucleic acid encodes a mouseimmunoglobulin light chain which comprises a variable region selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

In other embodiments, the isolated nucleic acid encodes a mouseimmunoglobulin heavy chain which comprises a variable region selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26.

The invention also relates to recombinant vectors (e.g., expressionvectors, including mammalian cell expression vectors) that comprise anucleic acid encoding the mouse monoclonal immunoglobulin (e.g., a mouseimmunoglobulin light chain and a mouse immunoglobulin heavy chain), themouse immunoglobulin light chain, or the mouse immunoglobulin heavychain of the invention. In some embodiments, the invention is arecombinant vector comprising a nucleic acid, or a pair of recombinantvectors comprising nucleic acids encoding a mouse monoclonalimmunoglobulin that comprises a light chain variable region selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, or a heavy chainvariable region selected from the group consisting of SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ IDNO: 26, or both such light chain variable region and heavy chainvariable region.

In other embodiments, the recombinant vector comprises a nucleic acidencoding a mouse immunoglobulin light chain, wherein the mouseimmunoglobulin light chain comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

In other embodiments, the recombinant vector comprises a nucleic acidencoding a mouse immunoglobulin heavy chain, wherein the mouseimmunoglobulin heavy chain comprises SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

In other embodiments, the recombinant vector comprises a nucleic acidencoding a mouse immunoglobulin light chain and a mouse immunoglobulinheavy chain, wherein the mouse immunoglobulin light chain and mouseimmunoglobulin heavy chain associate together to form a mouse monoclonalimmunoglobulin that has binding specificity for human CD52. In oneembodiment, the mouse immunoglobulin light chain comprises a variableregion selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and themouse immunoglobulin heavy chain comprises a variable region selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26.

In particular embodiments, the recombinant vector of the invention is anexpression vector, such as a mammalian cell expression vector. Incertain embodiments, the vector is a plasmid or a viral vector (e.g., anadenoviral or AAV vector).

The invention also relates to a host cell that comprises one or morenucleic acids encoding the mouse monoclonal immunoglobulin (mouseimmunoglobulin light chain and mouse immunoglobulin heavy chain), themouse immunoglobulin light chain or the mouse immunoglobulin heavy chainof the invention. For example, in some embodiments, the host cellcomprises a recombinant vector (e.g., expression vector, mammalian cellexpression vector) of the invention.

In some embodiments, the host cell comprises nucleic acid encoding amouse immunoglobulin light chain and a mouse immunoglobulin heavy chain,wherein the mouse immunoglobulin light chain and the mouseimmunoglobulin heavy chain associate together to form a mouse monoclonalimmunoglobulin that has binding specificity for human CD52 and whereinthe mouse immunoglobulin light chain comprises a variable regionselected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and/or the mouseimmunoglobulin heavy chain comprises a variable region selected from thegroup consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, or both.

In some embodiments, the host cell comprises nucleic acid encoding amouse immunoglobulin light chain, wherein the mouse immunoglobulin lightchain comprises a light chain variable region selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12 and SEQ ID NO: 13.

In some embodiments, the host cell comprises a nucleic acid encoding amouse immunoglobulin heavy chain, wherein the mouse immunoglobulin heavychain comprises a heavy chain variable region selected from the groupconsisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

The invention also relates to a method of preparing a mouse monoclonalimmunoglobulin comprising maintaining a host cell of the invention(e.g., a host cell that contains one or more recombinant nucleic acids(e.g., recombinant vectors) that encode a mouse monoclonalimmunoglobulin (e.g., a mouse immunoglobulin light chain and a mouseimmunoglobulin heavy chain) of the invention) under conditionsappropriate for expression of a mouse monoclonal immunoglobulin, wherebymouse monoclonal immunoglobulin chains are expressed and a mousemonoclonal immunoglobulin is produced. In some embodiments, the methodfurther comprises purifying or isolating the mouse monoclonalimmunoglobulin.

The invention also relates to a method of preparing a light chain of amouse monoclonal immunoglobulin, comprising maintaining a host cell ofthe invention containing a nucleic acid encoding a mouse immunoglobulinlight chain of the invention under conditions appropriate for expressionof said mouse immunoglobulin light chain, whereby a light chain isexpressed. In some embodiments, the method further comprises purifyingor isolating the light chain.

The invention also relates to a method of preparing a heavy chain of amouse monoclonal immunoglobulin, comprising maintaining a host cell ofthe invention containing a nucleic acid encoding a mouse immunoglobulinheavy chain of the invention under conditions appropriate for expressionof said mouse immunoglobulin heavy chain, whereby a mouse immunoglobulinheavy chain is expressed. In some embodiments, the method furthercomprises purifying or isolating the mouse immunoglobulin heavy chain.

The invention also relates to a method of diagnosing a disease (e.g.,autoimmune diseases (e.g., multiple sclerosis, lupus, vasculitis),cancer (e.g., leukemias (e.g., chronic lymphocytic leukemia), andlymphomas (e.g., non-Hodgkin's lymphoma)), and transplant (e.g., solidorgan transplant (e.g., kidney transplant) and stem cell transplant)')comprising assaying a patient sample in vitro, with the mouse monoclonalimmunoglobulin of the invention (e.g., Lundin, J., et al., Blood,101:4267-4272 (2003); Rodig, S J, et al., Clin. Cancer res., 12(23);7174-717179 (2006)).

Chimeric Immunoglobulins

The invention also relates to chimeric immunoglobulins that have bindingspecificity for human CD52. Such chimeric immunoglobulins may includethe variable regions of any of the mouse monoclonal immunoglobulin ofthe present invention. In one embodiment, the chimeric immunoglobulin ofthe invention comprises the light chain variable region of SEQ ID NO: 3and the heavy chain variable region of SEQ ID NO: 16; the light chainvariable region of SEQ ID NO: 4 and the heavy chain variable region ofSEQ ID NO: 17; the light chain variable region of SEQ ID NO: 5 and theheavy chain variable region of SEQ ID NO: 18; the light chain variableregion of SEQ ID NO: 6 and the heavy chain variable region of SEQ ID NO:19; the light chain variable region of SEQ ID NO: 7 and the heavy chainvariable region of SEQ ID NO: 20; the light chain variable region of SEQID NO: 8 and the heavy chain variable region of SEQ ID NO: 21; the lightchain variable region of SEQ ID NO: 9 and the heavy chain variableregion of SEQ ID NO: 22; the light chain variable region of SEQ ID NO:10 and the heavy chain variable region of SEQ ID NO: 23; the light chainvariable region of SEQ ID NO: 11 and the heavy chain variable region ofSEQ ID NO: 24; the light chain variable region of SEQ ID NO: 12 and theheavy chain variable region of SEQ ID NO: 25; or the light chainvariable region of SEQ ID NO: 13 and the heavy chain variable region ofSEQ ID NO: 26.

The invention also relates to a chimeric antibody that has bindingspecificity for human CD52, comprising a light chain variable regionsequence selected from the group consisting of the light chain variableregion of SEQ ID NO: 3, the light chain variable region of SEQ ID NO: 4,the light chain variable region of SEQ ID NO: 5, the light chainvariable region of SEQ ID NO: 6, the light chain variable region of SEQID NO: 7, the light chain variable region of SEQ ID NO: 8, the lightchain variable region of SEQ ID NO: 9, the light chain variable regionof SEQ ID NO: 10, the light chain variable region of SEQ ID NO: 11, thelight chain variable region of SEQ ID NO: 12 and the light chainvariable region of SEQ ID NO: 13, and/or a heavy chain variable regionsequence selected from the group consisting of: the heavy chain variableregion of SEQ ID NO: 16, the heavy chain variable region of SEQ ID NO:17, the heavy chain variable region of SEQ ID NO: 18, the heavy chainvariable region of SEQ ID NO: 19, the heavy chain variable region of SEQID NO: 20, the heavy chain variable region of SEQ ID NO: 21, the heavychain variable region of SEQ ID NO: 22, the heavy chain variable regionof SEQ ID NO: 23, the heavy chain variable region of SEQ ID NO: 24, theheavy chain variable region of SEQ ID NO: 25 and the heavy chainvariable region of SEQ ID NO: 26.

The invention also relates to a chimeric light chain comprising avariable region selected from the group consisting of SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

The invention also relates to a chimeric heavy chain comprising avariable region selected from the group consisting of SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ IDNO: 26.

Preferably, the chimeric immunoglobulins of the present inventioncomprise both a chimeric light chain of the invention and a chimericheavy chain of the invention.

In some embodiments, the invention provides a chimeric immunoglobulinwhich binds to the same epitope on human CD52 as a mouse monoclonalantibody comprising a light chain variable region of SEQ ID NO: 3 and aheavy chain variable region of SEQ ID NO: 16; a light chain variableregion of SEQ ID NO: 4 and a heavy chain variable region of SEQ ID NO:17; a light chain variable region of SEQ ID NO: 5 and a heavy chainvariable region of SEQ ID NO: 18; a light chain variable region of SEQID NO: 6 and a heavy chain variable region of SEQ ID NO: 19; a lightchain variable region of SEQ ID NO: 7 and a heavy chain variable regionof SEQ ID NO: 20; a light chain variable region of SEQ ID NO: 8 and aheavy chain variable region of SEQ ID NO: 21; a light chain variableregion of SEQ ID NO: 9 and a heavy chain variable region of SEQ ID NO:22; a light chain variable region of SEQ ID NO: 10 and a heavy chainvariable region of SEQ ID NO: 23; a light chain variable region of SEQID NO: 11 and a heavy chain variable region of SEQ ID NO: 24; a lightchain variable region of SEQ ID NO: 12 and a heavy chain variable regionof SEQ ID NO: 25; or a light chain variable region of SEQ ID NO: 13 anda heavy chain variable region of SEQ ID NO: 26. In other embodiments,the chimeric immunoglobulin binds to an epitope on human CD52 whichoverlaps with the epitope to which such a mouse monoclonal antibodybinds.

In other embodiments, the invention provides a chimeric immunoglobulinwhich binds to an epitope on human CD52 comprising at least residue 1 ofthe mature human CD52 sequence. The chimeric immunoglobulin may bind toan epitope comprising at least residues 1, 3, 4 and 5 of the maturehuman CD52 sequence, may bind to an epitope comprising at least residues1, 2, 3, 4 and 5 of the mature human CD52 sequence, or may bind to anepitope on human CD52 comprising at least residues 7, 8 and 9 of themature human CD52 sequence. In some embodiments, the epitope comprisesat least residues 7, 8 and 11 of the mature human CD52 sequence. In someembodiments, the epitope comprises at least residues 4 and 11 of themature human CD52 sequence.

The invention also relates to isolated nucleic acid molecules thatencode the chimeric immunoglobulins, chimeric light chains or chimericheavy chains of the invention. In some embodiments, the invention is anisolated nucleic acid molecule (one or more nucleic acid molecules)encoding a chimeric heavy chain and a chimeric light chain whichassociate together to form a chimeric immunoglobulin that has bindingspecificity for human CD52, wherein the chimeric light chain comprises avariable region selected from the group consisting of SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13;and/or the chimeric heavy chain comprises a variable region selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

In some embodiments, the invention is an isolated nucleic acid moleculeencoding a chimeric light chain that comprises the variable region ofSEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12or SEQ ID NO: 13.

In some embodiments, the invention is an isolated nucleic acid moleculeencoding a chimeric heavy chain that comprises the variable region ofSEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25 or SEQ ID NO: 26.

The invention also relates to recombinant vectors (e.g., expressionvectors, mammalian cell expression vectors) that comprise a nucleic acidencoding the chimeric immunoglobulin (chimeric light chain and chimericheavy chain), the chimeric light chain, or the chimeric heavy chain ofthe invention. In some embodiments, the invention is a recombinantvector comprising a nucleic acid (or a pair of recombinant vectorscomprising nucleic acids) encoding a chimeric immunoglobulin thatcomprises a light chain variable region selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12 and SEQ ID NO: 13; or a heavy chain variable regionselected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26; or bothsuch light chain and heavy chain.

In other embodiments, the recombinant vector comprises a nucleic acidencoding a chimeric light chain, wherein the chimeric light chaincomprises the variable region of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

In other embodiments, the recombinant vector comprises a nucleic acidencoding a chimeric heavy chain, wherein the chimeric heavy chaincomprises the variable region of SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

In particular embodiments, the recombinant vector of the invention is anexpression vector, such as a mammalian cell expression vector. Incertain embodiments, the vector is a plasmid or a viral vector (e.g., anadenoviral or AAV vector).

The invention also relates to a host cell that comprises one or morenucleic acids (e.g., one or more recombinant vectors) encoding thechimeric immunoglobulin (chimeric light chain and chimeric heavy chain),the chimeric light chain or the chimeric heavy chain of the invention.For example, in some embodiments, the host cell comprises a recombinantvector (e.g., expression vector, mammalian cell expression vector) ofthe invention.

In some embodiments, the host cell comprises a recombinant nucleic acid(or a pair of recombinant nucleic acids) encoding a chimeric light chainand a chimeric heavy chain, wherein the chimeric light chain and thechimeric heavy chain associate together to form a chimericimmunoglobulin that has binding specificity for human CD52 and whereinthe chimeric light chain comprises a variable region selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12 and SEQ ID NO: 13; and/or the chimeric heavy chaincomprises a variable region selected from the group consisting of thevariable region of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

In some embodiments, the host cell comprises a recombinant nucleic acidencoding a chimeric light chain, wherein the chimeric light chaincomprises a light chain variable region selected from the groupconsisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, and SEQ ID NO: 13.

In some embodiments, the host cell comprises a recombinant nucleic acidencoding a chimeric heavy chain, wherein the chimeric heavy chaincomprises a heavy chain variable region selected from the groupconsisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

The invention also relates to a method of preparing a chimericimmunoglobulin comprising maintaining a host cell of the invention(e.g., a host cell that contains one or more isolated nucleic acids thatencode a chimeric immunoglobulin (e.g., a chimeric light chain and achimeric heavy chain) of the invention) under conditions appropriate forexpression of a chimeric immunoglobulin, whereby chimeric immunoglobulinchains are expressed and a chimeric immunoglobulin is produced. In someembodiments, the method further comprises purifying or isolating thechimeric immunoglobulin.

The invention also relates to a method of preparing a chimeric lightchain comprising maintaining a host cell of the invention (e.g., a hostcell that contains a nucleic acid encoding a chimeric light chain of theinvention) under conditions appropriate for expression of said chimericlight chain, whereby a chimeric light chain is expressed and a chimericlight chain is produced. In some embodiments, the method furthercomprises purifying or isolating the chimeric light chain.

The invention also relates to a method of preparing a chimeric heavychain comprising maintaining a host cell of the invention (e.g., a hostcell that contains a nucleic acid encoding a chimeric heavy chain of theinvention) under conditions appropriate for expression of said chimericheavy chain, whereby a chimeric heavy chain is expressed and a chimericheavy chain is produced. In some embodiments, the method furthercomprises purifying or isolating the chimeric heavy chain.

The invention also relates to a method of diagnosing a disease selectedfrom the group consisting of autoimmune diseases (e.g., multiplesclerosis, lupus, vasculitis), cancer (e.g., leukemias (e.g., chroniclymphocytic leukemia), and lymphomas (e.g., non-Hodgkin's lymphoma)),and transplant (e.g., solid organ transplant (e.g., kidney transplant)and stem cell transplant’, comprising assaying a patient sample invitro, with the chimeric immunoglobulin of the invention.

Further embodiments of this invention are described as follows. In oneaspect, the invention relates to a monoclonal anti-human CD52 antibodyor an antigen-binding portion thereof, wherein the light chain and heavychain of said antibody comprise the three complementarity determiningregions (CDRs) found in: SEQ ID NOs: 3 and 16, respectively; SEQ ID NOs:4 and 17, respectively; SEQ ID NOs: 5 and 18, respectively; SEQ ID NOs:6 and 19, respectively; SEQ ID NOs: 7 and 20, respectively; SEQ ID NOs:8 and 21, respectively; SEQ ID NOs: 9 and 22, respectively; SEQ ID NOs:10 and 23, respectively; SEQ ID NOs: 11 and 24, respectively; SEQ IDNOs: 12 and 25, respectively; SEQ ID NOs: 12 and 137, respectively; orSEQ ID NOs: 13 and 26, respectively. In some embodiments, the inventionrelates to an antibody that binds to the same epitope on human CD52 asthe above monoclonal antibody or antigen-binding portion. In someembodiments, the invention relates to an antibody that competes with theabove monoclonal antibody or antigen-binding portion. In someembodiments, the invention relates to an antibody that cross-competeswith the above monoclonal antibody or antigen-binding portion.

In some embodiments, any of the above antibodies or antigen-bindingportions binds to an amino acid sequence comprising SEQ ID NO: 104. Insome related embodiments, the binding of said antibody or portion to SEQID NO: 104 may be reduced by an alanine substitution at one or more ofresidues 4, 7, 8, or 11 of SEQ ID NO: 104.

In some embodiments, the antibody is a humanized antibody, a mouseantibody, or a chimeric antibody. In certain embodiments, the frameworkregions of the heavy chain of said antibody utilize a VH3-72 or VH3-23human germline sequence, and the framework regions of the light chain ofsaid antibody utilize a VK2 A18b human germline sequence.

In some embodiments, the invention relates to a monoclonal anti-humanCD52 antibody or an antigen-binding portion thereof, wherein saidantibody comprises heavy chain (H)-CDR1, H-CDR2, H-CDR3, and light chain(L)-CDR1, L-CDR2, and L-CDR3 whose amino acid sequences are SEQ ID NOs:51, 59, 69, 29, 36, and 43, respectively; SEQ ID NOs: 50, 60, 69, 29,37, and 43, respectively; SEQ ID NOs: 50, 61, 68, 29, 38, and 43,respectively; SEQ ID NOs: 50, 61, 69, 29, 36, and 43, respectively; SEQID NOs: 50, 62, 69, 29, 39, and 43, respectively; SEQ ID NOs: 52, 61,70, 30, 40, and 43, respectively; SEQ ID NOs: 53, 63, 71, 31, 36, and44, respectively; SEQ ID NOs: 54, 64, 71, 31, 36, and 45, respectively;SEQ ID NOs: 55, 63, 72, 31, 36, and 46, respectively; SEQ ID NOs: 56,65, 73, 32, 41, and 47, respectively; SEQ ID NOs: 56, 65, 294, 32, 41,and 47, respectively; or SEQ ID NOs: 56, 66, 74, 33, 41, and 48,respectively.

In some embodiments, the invention relates to a monoclonal anti-humanCD52 antibody or an antigen-binding portion thereof, wherein the lightchain and heavy chain of said antibody comprise the amino acid sequencesof SEQ ID NOs: 3 and 16, respectively; SEQ ID NOs: 4 and 17,respectively; SEQ ID NOs: 5 and 18, respectively; SEQ ID NOs: 6 and 19,respectively; SEQ ID NOs: 7 and 20, respectively; SEQ ID NOs: 8 and 21,respectively; SEQ ID NOs: 9 and 22, respectively; SEQ ID NOs: 10 and 23,respectively; SEQ ID NOs: 11 and 24, respectively; SEQ ID NOs: 12 and25, respectively; or SEQ ID NOs: 13 and 26, respectively.

In some embodiments, the invention relates to a monoclonal antibody orantigen-binding portion thereof, wherein the heavy chain and light chainof said antibody comprise the amino acid sequences of SEQ ID NOs: 103and 102, respectively; SEQ ID NOs: 136 and 138, respectively; SEQ IDNOs: 137 and 138, respectively; SEQ ID NOs: 139 and 147, respectively;SEQ ID NOs: 149 and 155, respectively; SEQ ID NOs: 149 and 156,respectively; SEQ ID NOs: 158 and 165, respectively; SEQ ID NOs: 158 and166, respectively; SEQ ID NOs: 159 and 165, respectively; SEQ ID NOs:159 and 166, respectively; SEQ ID NOs: 161 and 166, respectively; or SEQID NOs: 163 and 166, respectively. In some embodiments, the inventionrelates to an antibody that binds to the same epitope on human CD52 asthe above monoclonal antibody or antigen-binding portion. In someembodiments, the invention relates to an antibody that competes with theabove monoclonal antibody or antigen-binding portion. In someembodiments, the invention relates to an antibody that cross-competeswith the above monoclonal antibody or antigen-binding portion.

In certain embodiments, the invention relates to a monoclonal humanizedanti-human CD52 antibody or an antigen-binding portion thereof, whereinthe heavy chain and the light chain of said antibody comprise the aminoacid sequences of SEQ ID NOs: 272 and 273, respectively, without thesignal sequences. In certain embodiments, the invention relates to amonoclonal anti-human CD52 antibody or an antigen-binding portionthereof, wherein the heavy chain and the light chain of said antibodycomprise the amino acid sequences of SEQ ID NOs: 274 and 275,respectively, without the signal sequences. In certain embodiments, theinvention relates to a monoclonal anti-human CD52 antibody or anantigen-binding portion thereof, wherein the heavy chain and the lightchain of said antibody comprise the amino acid sequences of SEQ ID NOs:276 and 278, respectively, without the signal sequences. In certainembodiments, the invention relates to a monoclonal anti-human CD52antibody or an antigen-binding portion thereof, wherein the heavy chainand the light chain of said antibody comprise the amino acid sequencesof SEQ ID NOs: 277 and 278, respectively, without the signal sequences.In certain embodiments, the invention relates to a monoclonal anti-humanCD52 antibody or an antigen-binding portion thereof, wherein the heavychain and the light chain of said antibody comprise the amino acidsequences of SEQ ID NOs: 279 and 280, respectively, without the signalsequences. In certain embodiments, the invention relates to a monoclonalanti-human CD52 antibody or an antigen-binding portion thereof, whereinthe heavy chain and the light chain of said antibody comprise the aminoacid sequences of SEQ ID NOs: 281 and 282, respectively, without thesignal sequences. The invention also provides antibodies that bind tothe same epitope on CD52 as one of these humanized antibodies andantibodies that compete or cross-compete with one of these humanizedantibodies. In related embodiments, the invention provides compositionscomprising one such humanized antibody and a pharmaceutically acceptablecarrier.

In some embodiments, the invention relates to a monoclonal anti-humanCD52 antibody or an antigen-binding portion thereof, wherein the lightchain of said antibody comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 102, 138, 145-148, 153-157, and164-168. In certain embodiments, the invention relates to a monoclonalanti-human CD52 antibody or an antigen-binding portion thereof, whereinthe light chain of said antibody comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 273, 275, 278, 280,and 282, without the signal sequences. In certain embodiments, theinvention relates to an antibody light chain or a portion thereof,comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 102, 138, 145-148, 153-157, 164-168, 273, 275, 278, 280, and282, without the signal sequences if present.

In some embodiments, the invention relates to a monoclonal anti-humanCD52 antibody or an antigen-binding portion thereof, wherein the heavychain of said antibody comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 103, 136, 137, 139-144, 149-152, and158-163. In certain embodiments, the invention relates to a monoclonalanti-human CD52 antibody or an antigen-binding portion thereof, whereinthe heavy chain of said antibody comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 272, 274, 276, 277,279, and 281, without the signal sequences. In certain embodiments, theinvention relates to an antibody heavy chain or a portion thereof,comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 103, 136, 137, 139-144, 149-152, 158-163, 272, 274, 276,277, 279, and 281, without the signal sequences if present.

In some embodiments, any of the above antibodies may be an IgG, IgM,IgA, IgD or IgE molecule. In certain embodiments, said IgG is IgG1,IgG2, IgG3, or IgG4.

In some embodiments, any of the above antigen-binding portions may be asingle chain antibody, Fv, Fab, Fab′, F(ab′)2, Fd, single chain Fvmolecule (scFv), bispecific single chain Fv dimer, diabody,domain-deleted antibody or single domain antibody (dAb).

The invention also relates to any of the above antibodies orantigen-binding portions, wherein said antibody or antigen-bindingportion depletes T or B lymphocytes, or both; preferentially depletes Tlymphocytes as compared to B lymphocytes; increases circulating serumlevels of TNF-alpha, IL-6, or MCP-1 (e.g., by at least 5%, at least 10%,at least 50%, at least 100% or at least 200%); mediatesantibody-dependent cell mediated cytotoxicity (ADCC) of CD52-expressingcells; mediates complement-dependent cytotoxicity (CDC) ofCD52-expressing cells; binds to human CD52 in spite of the presence ofneutralizing antibodies to alemtuzumab in a human patient; and/orpromotes intracellular signaling in human T and/or B cells (see, e.g.,Hederer et al., International Immunology 12:505-616 (2000); Watanabe etal., Clinical Immunology 120: 247-259 (2006)).

The invention further relates to an isolated nucleic acid encoding theheavy chain or an antigen-binding portion thereof, or the light chain oran antigen-binding portion thereof, of any of the above antibodies. Insome embodiments, said isolated nucleic acid comprises a heavy chainnucleotide sequence selected from the group consisting of SEQ ID NOs:283, 285, 287, 288, 290, and 292, or said nucleotide sequence withoutthe sequence encoding a signal peptide; a light chain nucleotidesequence selected from the group consisting of SEQ ID NOs: 284, 286,289, 291, and 293, or said nucleotide sequence without the sequenceencoding a signal peptide; or both said heavy chain nucleotide sequenceand said light chain nucleotide sequence. In certain embodiments, saidisolated nucleic acid comprises a heavy chain nucleotide sequence and alight chain nucleotide sequence selected from the group consisting ofSEQ ID NO: 283 and SEQ ID NO: 284, respectively, both without sequencesencoding signal peptides; SEQ ID NO: 285 and SEQ ID NO: 286,respectively, both without sequences encoding signal peptides; SEQ IDNO: 287 and SEQ ID NO: 289, respectively, both without sequencesencoding signal peptides; SEQ ID NO: 288 and SEQ ID NO: 289,respectively, both without sequences encoding signal peptides; SEQ IDNO: 290 and SEQ ID NO: 291, respectively, both without sequencesencoding signal peptides; and SEQ ID NO: 292 and SEQ ID NO: 293,respectively, both without sequences encoding signal peptides.

The invention also relates to the use of an isolated nucleic acidcomprising a heavy chain nucleotide sequence and an isolated nucleicacid comprising a light chain nucleotide sequence for the manufacture ofa medicament for treating a patient in need thereof, wherein said heavychain nucleotide sequence and light chain nucleotide sequence areselected from the group consisting of SEQ ID NO: 283 and SEQ ID NO: 284,respectively, both without sequences encoding signal peptides; SEQ IDNO: 285 and SEQ ID NO: 286, respectively, both without sequencesencoding signal peptides; SEQ ID NO: 287 and SEQ ID NO: 289,respectively, both without sequences encoding signal peptides; SEQ IDNO: 288 and SEQ ID NO: 289, respectively, both without sequencesencoding signal peptides; SEQ ID NO: 290 and SEQ ID NO: 291, bothrespectively, without sequences encoding signal peptides; and SEQ ID NO:292 and SEQ ID NO: 293, both respectively, without sequences encodingsignal peptides.

The invention also relates to a recombinant vector comprising (1) anucleic acid sequence encoding the heavy chain or an antigen-bindingportion thereof, (2) a nucleic acid sequence encoding the light chain oran antigen-binding portion thereof, or (3) both, of any of the aboveantibodies. The invention further relates to a host cell comprising afirst nucleic acid sequence encoding the heavy chain or anantigen-binding portion thereof of any of the above antibodies, saidfirst nucleic acid sequence operably linked to an expression controlelement, and a second nucleic acid sequence encoding the light chain oran antigen-binding portion thereof of said antibody, said second nucleicacid sequence operably linked to an expression control element. Theinvention relates to a method of making an anti-human CD52 antibody oran antigen-binding portion thereof, comprising maintaining said hostcell under conditions appropriate for expression of the antibody orportion, and also relates to said method further comprising the step ofisolating the antibody or portion.

The invention relates to a composition comprising the monoclonalantibody or antigen-binding portion according to any one of claims 1-24and a pharmaceutically acceptable vehicle or carrier.

In some embodiments, the invention relates to a method for treating apatient in need thereof, comprising administering to the patient aneffective amount of any of the above antibodies or antigen-bindingportions, or the above composition. In certain embodiments, said patientis receiving a transplantation.

In some embodiments, the invention relates to a method for treating anautoimmune disease in a patient in need thereof, comprisingadministering to the patient an effective amount of any of the aboveantibodies or antigen-binding portions, or the above composition. Incertain embodiments, the autoimmune disease is, e.g., multiplesclerosis, rheumatoid arthritis, or systemic lupus erythematosus.

In some embodiments, the invention relates to a method for treatingcancer in a patient in need thereof, comprising administering to thepatient an effective amount of any of the above antibodies orantigen-binding portions, or the above composition. In certainembodiments, the cancer is, e.g., a lymphoma such as non-Hodgkin'slymphoma; a leukemia such as B-cell chronic lymphocytic leukemia; T cellmalignancy, wherein the antibody or portion preferentially depletes Tcells as compared to B cells; or a solid tumor.

In some embodiments, any of the above methods of treatment furthercomprising administering to the patient a neutrophil or NK cellstimulatory agent. In certain embodiments, said agent is G-CSF orGM-CSF. In some embodiments, any of the above methods of treatmentfurther comprises administering to the patient a T regulatory cellstimulatory agent. In certain embodiments, said agent is rapamycin.

In some embodiments, the invention relates to a method for inhibitingangiogenesis in a patient in need thereof, comprising administering aneffective amount of any of the above antibodies or antigen-bindingportions to the patient. In certain embodiments, the patient has a solidtumor. In certain embodiments, the patient has neovascularization. Incertain embodiments, said neovascularization is in the eye.

The invention also relates to the use of any of the above antibodies orantigen-binding portions for the manufacture of a medicament fortreating an autoimmune disease in a patient in need thereof. Further,the invention relates to the use of any of the above antibodies orantigen-binding portions for the manufacture of a medicament fortreating cancer in a patient in need thereof. The invention relates tothe use of any of the above antibodies or antigen-binding portions forthe manufacture of a medicament for treating a patient in need of atransplantation. The invention relates to the use of any of the aboveantibodies or antigen-binding portions for the manufacture of amedicament for treating neovascularization in a patient in need thereof.

The invention also relates to the use of any of the above antibodies orantigen-binding portions as a medicament.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B is a schematic representation of the development of newanti-CD52 monoclonal antibodies. The general scheme is depicted in FIG.1A and the names of the mouse anti-human CD52 antibody clones as well astheir isotypes in shown in FIG. 1B.

FIG. 2 is an alignment of the amino acid sequences of several mouseanti-human CD52 kappa light chain sequences (SEQ ID NOS:1-13).Campath-1G is the rat monoclonal antibody from which the humanizedCampath-1H antibody is derived.

FIG. 3 is an alignment of the amino acid sequences of several mouseanti-human CD52 heavy chain sequences (SEQ ID NOS:14-26).

FIG. 4 is an alignment of wildtype CD52 and 10 mutant CD52 proteins (SEQID NOS: 104-114, from top to bottom).

FIG. 5A illustrates the FACS-based N-terminal binding profile ofantibodies 4B10 and 7F11 on cells expressing CD52 alanine scanningmutants.

FIG. 5B illustrates the FACS-based middle region binding profile ofantibodies CF1D12, 3G7, 9D9, 5F7, 4G7, and 11C11 on cells expressingCD52 alanine scanning mutants.

FIG. 5C illustrates the FACS-based binding profile of antibodiesCampath-1H® (“Campath 1H”), 2C3, 12G6, and 23E6 on cells expressing CD52alanine scanning mutants.

FIG. 5D depicts immunoblots of CD52+/− N-linked glycosylation probedwith the panel of chimeric monoclonal antibodies. “C1H” stands forCampath-1H®.

FIG. 6 is a graph showing the results of a 1.5 hour CDC assay on variouschimeric anti-CD52 antibodies screened on CHO-K1 CD52 #67 cells. Theresults show that chimeric antibodies 4B10 and 7F11 are comparable to orbetter than Campath-1H® (“Campath 1H”).

FIG. 7 is a graph showing the results of a 14 hour ADCC assay on variouschimeric IgG1 antibodies to CD52 screened on CHO-K1 CD52 #67 cells. Theresults show that chimeric antibodies 2C3 and 12G6 are comparable to orbetter than Campath-1H® (“Campath 1-H”).

FIG. 8A-8C illustrate the comparative binding of various anti-CD52antibodies and the Campath-1H® (“C-1H”) antibody to defined humanlymphocyte populations. These figures show the hierarchy of the bindingability of the chimeric antibodies screened by FACS assay. Curves to thefar right demonstrate the highest binding ability, whereas curves to theleft bind with lower affinity.

FIGS. 9A-9C are graphs illustrating the level of CD4 T cells (FIG. 9A),CD8 T cells (FIG. 9B) and CD19 B cells (FIG. 9C) in the blood 72 hoursafter dosing with chimeric antibodies 7F11, 8G3, 23E6, 12G6, 4B10, or5F7, or Campath-1H® (“Cam”).

FIGS. 10A-10C are graphs illustrating the level of CD4 T cells (FIG.10A), CD8 T cells (FIG. 10B) and CD19 B cells (FIG. 10C) in the spleen72 hours after dosing with chimeric antibodies 7F11, 8G3, 23E6, 12G6,4B10, or 5F7, or Campath-1H® (“Cam”).

FIGS. 11A-11C are graphs showing the level of CD4 T cells (FIG. 11A),CD8 T cells (FIG. 11B) and CD19 B cells (FIG. 11C) in the blood 72 hoursafter dosing with chimeric antibodies 2C3, 9D9, 4B10, 3G7, or 11C11, orCampath-1H® (“Cam”).

FIG. 12 is a Kaplan Meier Survival graph illustrating the percent ofsurviving mice after treatment with 7F11, 4B10, or 12G6 chimericmonoclonal antibodies, or Campath-1H® (“Campath”).

FIG. 13 is a Kaplan Meier Survival graph illustrating the percent ofsurviving mice after treatment with 2C3, 8G3, or 23E6 chimericmonoclonal antibodies, or Campath-1H® (“Campath”).

FIG. 14 is a Kaplan Meier Survival graph illustrating the percent ofsurviving mice after treatment with 9D9 or 4B10 chimeric monoclonalantibodies, or Campath-1H® (“Campath”).

FIG. 15 is a Kaplan Meier Survival graph illustrating the percent ofsurviving mice after treatment with 2C3 or 11C11 chimeric monoclonalantibodies, or Campath-1H® (“Campath”).

FIG. 16 is an alignment of the mouse anti-human CD52 antibody 4B10 heavychain variable region (SEQ ID NO: 96) sequence with the closest matchedhuman germline sequence (SEQ ID NO: 97) and the humanized heavy chainvariable region sequence (SEQ ID NO: 98). Also shown is an alignment ofthe mouse anti-human CD52 antibody 4B10 light chain variable region (SEQID NO: 99) sequence with the closest matched human germline sequence(SEQ ID NO: 100) and the humanized light chain variable region sequence(SEQ ID NO: 101).

FIG. 17 shows the humanized 4B10 heavy chain (SEQ ID NO: 103) and lightchain (SEQ ID NO: 102) variable region sequences.

FIG. 18 is a graph showing that humanized antibody 4B10-H1/K1(“4B10-Humanized”) and chimeric antibody 4B10 bind equivalently to cellsexpressing CD52.

FIG. 19 is a graph showing that humanized antibody 4B10-H1/K1 (“4B10Humanized”) and chimeric antibody 4B10 mediate equivalent ADCC activityon cells expressing CD52.

FIG. 20 is a graph showing that humanized antibody 4B10-H1/K1(“4B10-Humanized”) and chimeric antibody 4B10 mediate equivalent CDCactivity on cells expressing CD52.

FIG. 21 is a graph illustrating the pharmacokinetic profile of chimericanti-CD52 antibodies (12G6, 7F11 and 4B10), Campath-1H® (“Campath”), andhumanized anti-CD52 antibody 4B10-H1/K1 (“4B10 humanized (H1/K1)”) inheterozygous huCD52 transgenic mice.

FIGS. 22A-22C are graphs showing the level of CD4 T cells (FIG. 22A),CD8 T cells (FIG. 22B) and CD19 B cells (FIG. 22C) in the blood 72 hoursafter dosing with chimeric antibody 4B10 or humanized antibody4B10-H1/K1 (“4B10-Hu”) or Campath-1H® (“Campath”).

FIG. 23 is a graph showing the summary of the relative bindingaffinities of the anti-CD52 monoclonal antibodies.

FIG. 24 shows the humanized 7F11 heavy and light (kappa) chain variableregion sequences. Amino acid residues that are back mutated to mouseresidues are underlined and the CDRs are shown in boldface.

FIG. 25 is a histogram showing that chimeric and humanized 7F11antibodies bind equivalently to cells expressing CD52. The X axisrepresents the fluorescence emitted by the bound anti-CD52 antibody,while the area of each peak represents the total cell population.

FIG. 26A shows the humanized 2C3 heavy chain variable region sequences.Amino acid residues that are back mutated to mouse residues areunderlined and the CDRs are shown in boldface. FIG. 26B shows thehumanized 2C3 light (kappa) chain variable region sequences. Amino acidresidues that are back mutated to mouse residues are underlined and theCDRs are shown in boldface.

FIG. 27A is a histogram showing binding of humanized and chimeric 2C3antibodies to cells expressing CD52. The X axis represents thefluorescence emitted by the bound anti-CD52 antibody, while the area ofeach peak represents the total cell population. FIG. 27B is a histogramshowing that chimeric and a subset of the humanized 2C3 antibodies bindequivalently to cells expressing CD52. The X axis represents thefluorescence emitted by the bound anti-CD52 antibody, while the area ofeach peak represents the total cell population.

FIG. 28A shows the humanized 12G6 heavy chain variable region sequences.Amino acid residues that are back mutated to mouse residues areunderlined and the CDRs are shown in boldface. FIG. 28B shows thehumanized 12G6 light (kappa) chain variable region sequences. Amino acidresidues that are back mutated to mouse residues are underlined and theCDRs are shown in boldface.

FIG. 29 is a histogram showing that chimeric and a subset of thehumanized 12G6 antibodies bind equivalently to cells expressing CD52.The X axis represents the fluorescence emitted by the bound anti-CD52antibody, while the area of each peak represents the total cellpopulation.

FIG. 30A shows the humanized 9D9 heavy chain variable region sequences.Amino acid residues that are back mutated to mouse residues areunderlined and the CDRs are shown in boldface. FIG. 30B shows thehumanized 9D9 light (kappa) chain variable region sequences. Amino acidresidues that are back mutated to mouse residues are underlined and theCDRs are shown in boldface.

FIG. 31 is a histogram showing that chimeric and a subset of thehumanized 9D9 antibodies bind equivalently to cells expressing CD52. TheX axis represents the fluorescence emitted by the bound anti-CD52antibody, while the area of each peak represents the total cellpopulation.

FIG. 32A shows the binding curves of Campath-1H® (“C1H”), a chimeric 2C3antibody, and a humanized 2C3-SFD1/K12 antibody to primary human T cellsand huCD52 transgenic mouse T cells. FIG. 32B shows the binding curvesof Campath-1H® (“C1H”), a chimeric 9D9 antibody, and humanized 9D9antibodies to primary human T cells and huCD52 transgenic mouse T cells.FIG. 32C shows the binding curves of Campath-1H® (“C1H”), a chimeric12G6 antibody, and humanized 12G6 antibodies to primary human T cellsand huCD52 transgenic mouse T cells.

FIG. 33 is a table showing the relative binding efficiency ofCampath-1H®, chimeric 2C3 and 12G6 antibodies, and humanized 2C3 and12G6 antibodies to huCD52 expressing human and transgenic mouse T cells.

FIG. 34 illustrates the comparative binding patterns of humanizedanti-CD52 Campath-1H®, 2C3, 12G6, and 9D9 antibodies to defined subsetsof human peripheral blood mononuclear cell populations by flowcytometry. These histograms show that the humanized anti-CD52 antibodybinding is equivalent to that of Campath-1H® for various CD52 expressinghuman PBMC subsets. The X axis represents the fluorescence emitted bythe bound anti-CD52 antibody, while the area of each peak represents thetotal cell population.

FIG. 35 is a graph showing that chimeric and humanized 7F11 antibodiesmediate equivalent ADCC activity on cells expressing CD52.

FIG. 36 is a graph showing that chimeric and humanized 7F11 antibodiesmediate CDC activity on cells expressing CD52.

FIG. 37 is a graph showing that chimeric and humanized 2C3 antibodiesmediate ADCC activity on cells expressing CD52.

FIG. 38 is a graph showing that chimeric and humanized 2C3 antibodiesmediate CDC activity on cells expressing CD52.

FIG. 39 is a graph showing that chimeric and humanized 12G6 antibodiesmediate ADCC activity on cells expressing CD52.

FIG. 40 is a graph showing that chimeric and humanized 12G6 antibodiesmediate CDC activity on cells expressing CD52.

FIG. 41 is a graph showing that chimeric and humanized 9D9 antibodiesmediate ADCC activity on cells expressing CD52.

FIG. 42 is a graph showing that chimeric and humanized 9D9 antibodiesmediate CDC activity on cells expressing CD52.

FIG. 43 is a graph showing the ADCC activity of humanized anti-CD52antibodies on primary T cells.

FIG. 44 is a graph showing the CDC activity of humanized anti-CD52antibodies on primary T cells.

FIG. 45 is a graph showing neutralization of Campath-1H but not otheranti-CD52 antibodies with CAMMS223 study human serum samples thatcontain anti-Campath-1H® neutralizing antibodies. Serum samples weretaken from a representative patient (MS-1) at month 12 (M12) and month13 (M13).

FIGS. 46A-46E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, myeloid cells, and neutrophils in the blood 72 hoursafter dosing with Campath-1H® (“Campath”) and humanized 4B10-H1/K1(“4B10”) antibodies.

FIGS. 47A-47E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, myeloid cells, neutrophils, and macrophages in thespleen 72 hours after dosing with Campath-1H® (“Campath”) and humanized4B10-H1/K1 (“4B10”) antibodies.

FIGS. 48A-48E show the levels of circulating cytokines 2 hours afterdosing with Campath-1H® (“Campath”) and humanized 4B10-H1/K1 (“4B10”)antibodies.

FIGS. 49A and 49B show the repopulation of circulating lymphocytes overa time course after dosing with Campath-1H® (“Campath”) and humanized4B10-H1/K1 (“4B10”) antibodies, (mg/kg).

FIGS. 50A-50E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, myeloid cells, and neutrophils in the blood 72 hoursafter dosing with the humanized 7F11-SFD1/K2 (“7F11 SFD1”) and7F11-SFD2/K2 (“7F11 SFD2”) antibodies.

FIGS. 51A-51E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, myeloid cells, and neutrophils in the spleen 72 hoursafter dosing with the humanized 7F11-SFD1/K2 (“7F11 SFD1”) and7F11-SFD2/K2 (“7F11 SFD2”) antibodies.

FIGS. 52A-52F show the levels of circulating cytokines 2 hours afterdosing with the humanized 7F11-SFD1/K2 (“7F11 SFD1”) and 7F11-SFD2/K2(“7F11 SFD2”) antibodies.

FIGS. 53A and 53B show the repopulation of circulating lymphocytes overa timecourse after dosing with the humanized 7F11-SFD1/K2 (“7F11 SFD1”)and 7F11-SFD2/K2 (“7F11 SFD2”) antibodies, (mg/kg).

FIGS. 54A and 54B show the level of CD4+ T cells, CD8+ T cells and B220+B cells in the blood 72 hours after dosing with Campath-1H® (“Campath”),7F11-chimeric antibodies, and humanized 7F11-SFD1/K2 and 7F11-SFD2/K2antibodies.

FIG. 55 shows the level of Campath-1H® (“Campath”), 7F11-chimericantibody and humanized 7F11-SFD1/K2 and 7F11-SFD2/K2 antibodies in theblood over a timecourse after dosing.

FIGS. 56A-56E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, and neutrophils in the blood 72 hours after dosing with2C3-SFD1/K12 antibodies.

FIGS. 57A-57E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, and neutrophils in the spleen 72 hours after dosingwith 2C3-SFD1/K12 antibodies.

FIGS. 58A-58F show the levels of circulating cytokines 2 hours afterdosing with 2C3-SFD1/K12 (“2C3”) antibodies.

FIG. 59 shows the repopulation of circulating lymphocytes over atimecourse after dosing with 2C3-SFD1/K12 antibodies, (mg/kg).

FIGS. 60A-60E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, myeloid cells, macrophages, and neutrophils in theblood 72 hours after dosing with 12G6-SFD1/K11 antibodies.

FIGS. 61A-61E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, macrophages, neutrophils, and myeloid cells in thespleen 72 hours after dosing with 12G6-SFD1/K11 antibodies.

FIGS. 62A-62F show the levels of circulating cytokines 2 hours afterdosing with 12G6-SFD1/K11 (“12G6 hu”) antibodies.

FIG. 63 shows the repopulation of circulating lymphocytes over atimecourse after dosing with 12G6-SFD1/K11 antibodies, (mg/kg).

FIGS. 64A-64C show the level of 2C3-chimeric, 2C3-SFD1/K12,12G6-chimeric, 12G6-SFD1/K11, 9D9-chimeric, and 9D9-H10/K12 antibodiesin the blood over a timecourse after dosing.

FIGS. 65A-65E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, myeloid cells, macrophages, and neutrophils in theblood 72 hours after dosing with 9D9-H10/K12 (“9D9”) antibodies.

FIGS. 66A-66E show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, myeloid cells, neutrophils, and macrophages in thespleen 72 hours after dosing with 9D9-H10/K12 (“9D9”) antibodies.

FIGS. 67A-67F show the levels of circulating cytokines 2 hours afterdosing with 9D9-H10/K12 (“9D9”) antibodies.

FIG. 68 shows the repopulation of circulating lymphocytes over atimecourse after dosing with 9D9-H10/K12 (“9D9”) antibodies, (mg/kg).

FIGS. 69A-69D show the level of bulk lymphocyte populations (CD4+ Tcells, CD8+ T cells, and B cells) and CD4+ T cell, CD8+ T cell, B220+ Bcell and NK cell subtypes in the blood 72 hours after dosing withCampath-1H® (“Campath”), 2C3-SFD1/K12 (“2C3”), 12G6-SFD1/K11 (“12G6”),and 9D9-H10/K12 (“9D9”) antibodies.

FIGS. 70A-70D show the level of bulk lymphocyte populations (CD4+ Tcells, CD8+ T cells, and B cells) and CD4+ T cell, CD8+ T cell, B220+ Bcell and NK cell subtypes in the spleen 72 hours after dosing withCampath-1H® (“Campath”), 2C3-SFD1/K12 (“2C3”), 12G6-SFD1/K11 (“12G6”),and 9D9-H10/K12 (“9D9”) antibodies.

FIGS. 71A-71F show the levels of circulating cytokines 2 hours afterdosing with Campath-1H®, 2C3-SFD1/K12, 12G6-SFD1/K11, and 9D9-H10/K12antibodies.

FIG. 72 shows the level of CD4+ T cells, CD8+ T cells, B220+ B cells,and NK cells in the blood 72 hours after dosing with 9D9-H10/K12 and9D9-H11/K12 antibodies.

FIG. 73 shows the level of CD4+ T cells, CD8+ T cells, B220+ B cells,and NK cells in the spleen 72 hours after dosing with 9D9-H10/K12 and9D9-H11/K12 antibodies.

FIGS. 74A-74D show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells, and myeloid cells in the blood 72 hours after dosingwith 12G6-SFD1/K11 (“12G6 K11”) and 12G6-SFD1/K12 (“12G6 K12”)antibodies.

FIGS. 75A-75D show the level of CD4+ T cells, CD8+ T cells, B220+ Bcells, NK cells and myeloid cells in the spleen 72 hours after dosingwith 12G6-SFD1/K11 (“12G6 K11”) and 12G6-SFD1/K12 (“12G6 K12”)antibodies.

FIG. 76 shows the level of bulk lymphocyte populations (CD4+ T cells,CD8+ T cells, and B220+ B cells) in the blood 72 hours after dosing with9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies.

FIGS. 77A-77D show the level of CD4+ T cell, CD8+ T cell, B220+ B cell,NK cell, and myeloid cell subtypes in the blood 72 hours after dosingwith 9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies.

FIG. 78 shows the level of bulk lymphocyte populations (CD4+ T cells,CD8+ T cells, and B220+ B cells) in the spleen 72 hours after dosingwith 9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies.

FIGS. 79A-79D show the level of CD4+ T cell, CD8+ T cell, B220+ B cell,NK cell, and myeloid cell subtypes in the spleen 72 hours after dosingwith 9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies.

FIGS. 80A-80F show the levels of circulating cytokines 2 hours afterdosing with 9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies.

FIGS. 81A and 81B show the level of 2C3-SFD1/K12, 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13 and 9D9-H18/K13 antibodies in the blood overa timecourse after dosing.

FIGS. 82A-82F show the level of cytokines in the blood over a 48-hourtimecourse following dosing with Campath-1H® (“Campath”), 2C3-SFD1/K11,12G6-SFD1/K11, 12G6-SFD1/K12, 9D9-H16/K13 or 9D9-H18/K13 antibodies.

FIGS. 83A-83E show the level of bulk lymphocytes, CD4+ T cells, CD8+ Tcells, B220+ B cells, NK cells, and myeloid cells in the spleen 72 hoursafter dosing with Campath-1H® (“Campath”), 2C3-SFD1/K11, 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13 or 9D9-H18/K13 antibodies.

FIGS. 84A-84G show the repopulation of circulating CD4+ and CD8+ Tcells, regulatory T cells, B cells, NK cells, neutrophils andmacrophages over a timecourse after dosing with Campath-1H® (“Campath”),2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12 antibodies.

FIG. 85 shows the ability of FITC-labeled Campath-1H® (“Campath”),2C3-SFD1/K12 (“2C3 K12”), 12G6-SFD1/K11 (“12G6 K11”), 12G6-SFD1/K12(“12G6 K12”), 9D9-H16/K13 (“9D9 H16”), and 9D9-H18/K13 (“9D9 H18”)antibodies to specifically bind huCD52 lymphocyte cell populations inthe spleen.

FIGS. 86A-86E show the level of bulk lymphocyte populations (CD4+ Tcells, CD8+ T cells, and B220+ B cells) and CD4+ T cell, CD8+ T cell,B220+ B cell, NK cell, and myeloid cell subtypes in the blood 72 hoursafter dosing with Campath-1H® (“Campath”), 2C3-SFD1/K12, 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies.

FIGS. 87A-87E show the level of bulk lymphocyte populations (CD4+ Tcells, CD8+ T cells, and B220+ B cells) and CD4+ T cell, CD8+ T cell,B220+ B cell, NK cell, and myeloid cell subtypes in the spleen 72 hoursafter dosing with Campath-1H® (“Campath”), 2C3-SFD1/K12, 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies.

FIGS. 88A-88F show the levels of circulating cytokines 2 hours afterdosing with Campath-1H® (“Campath”), 2C3-SFD1/K12, 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies.

FIGS. 89A-89D show the level of CD4+ T cell, CD8+ T cell, B220+ B cell,and NK/myeloid cell subtypes in the blood 72 hours after dosing withCampath-1H® (“Campath”), 2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12antibodies.

FIGS. 90A-90D show the level of CD4+ T cell, CD8+ T cell, B220+ B cell,and NK/myeloid cell subtypes in the spleen 72 hours after dosing withCampath-1H® (“Campath”), 2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12antibodies.

FIGS. 91A-91D show the level of CD4+ T cell, CD8+ T cell, B220+ B cell,and NK/myeloid cell subtypes in the lymph node 72 hours after dosingwith Campath-1H® (“Campath”), 2C3-SFD1/K12, 9D9-H16/K13 and12G6-SFD1/K12 antibodies.

FIG. 92A shows the huCD52 expression level on CD4+ T cell, CD8+ T cell,B220+ B cell, and NK/myeloid cell subtypes in huCD52-KI/KO andnon-transgenic control mice. FIG. 92B shows the huCD52 expression levelon CD4+ T cells, CD8+ T cells, and B cells in huCD52-KI/KO and huCD52CD1 transgenic mice.

FIG. 93 shows the binding to huCD52 of 12G6-SFD1/K12 and 2C3-SFD1/K12antibodies from various production sources (“small scale” and “largescale”) as compared to a Campath-1H® control.

FIG. 94 shows the level of bulk lymphocyte populations (CD4+ T cells,CD8+ T cells, B220+ B cells and NK cells) in the blood 72 hours afterdosing with 12G6-SFD1/K12 and 2C3-SFD1/K12 antibodies from variousproduction sources (“small scale” and “large scale”).

FIG. 95 shows the levels of 2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12antibodies in the blood over a timecourse after dosing.

FIG. 96 demonstrates the EAE clinical score of 2C3-SFD1/K12 and12G6-SFD1/K12 over a timecourse of disease progression.

FIGS. 97A and 97B demonstrate the ability of Campath1H® (“Campath”),2C3-SFD1/K12 (“2C3”), His435Ala 2C3-SFD1/K12 (“H435A 2C3”) andHis310Ala/His435Gln 2C3-SFD1/K12 (“H310A/H435Q 2C3”) to bind to mouseand human FcRn molecules.

FIG. 98 shows the in vivo clearance of 2C3-SFD1/K12 (“2C3 unmodified”),2C3-SFD1/K12-Modified 1 (“2C3-Fc mutant 1”) and 2C3-SFD1/K12-Modified 2(“2C3-Fc mutant 2”) in nontransgenic mice.

FIG. 99 shows the in vivo clearance of 2C3-SFD1/K12 (“2C3”),2C3-SFD1/K12-Modified 1 (“2C3-Fc mutant 1”) and 2C3-SFD1/K12-Modified 2(“2C3-Fc mutant 2”) in huCD52 transgenic mice.

FIGS. 100A and 100B show the level of bulk lymphocyte populations (CD4+T cells, CD8+ T cells, B220+ B cells, and NK cells) in the blood andspleen 72 hours after dosing with 2C3-SFD1/K12 (“2C3”),2C3-SFD1/K12-Modified 1 (“2C3 Fc mutant-1”), and 2C3-SFD1/K12-Modified 2(“2C3 Fc mutant-2”) antibodies.

FIGS. 101A and 101B are representative sensorgrams of Biacore T100assays to determine the epitope specificity of the humanized12G6-SFD1/K12 antibody and mutant peptides generated by alaninescanning. FIG. 101A shows no binding between 12G6-SFD1/K12 and the MUT 8peptide, while FIG. 101B shows binding between 12G6-SFD1/K12 and the MUT9 peptide.

FIG. 102 shows the TCR V beta analysis for donor BMS486. CD4+ T cellseducated with Campath-1H® peptide group 986-989 exhibited preferentialexpansion of a single V beta (Vβ3).

FIG. 103 shows the TCR V beta analysis for donor BMS928. CD4+ T cellseducated with 12G6-SFD1/K12 peptide groups 1066-67-68 and 1083-84-85exhibited preferential expansion of a single V beta (Vβ20).

FIGS. 104A-104J show the Campath-1H® immunogenicity assessment.Proliferative responses are shown in CPM for individual donors A-J. TheX axis depicts the groups of peptides used to stimulate autologous CD4+T cells three times. Each group of T cells was assayed in triplicatewith autologous DCs pulsed with the educating antigen/peptide group(specific response, left bar, white), irrelevant DR binding peptide(middle bar, striped), or media (right bar, black).

FIGS. 105A-105J show the 12G6-SFD1/K12 immunogenicity assessment.Proliferative responses are shown in CPM for individual donors A-J. TheX axis depicts the groups of peptides used to stimulate autologous CD4+T cells three times. Each group of T cells was assayed in triplicatewith autologous DCs pulsed with the educating peptide group (specificresponse, left bar, white), irrelevant DR binding peptide (middle bar,striped), or media (right bar, black). In groups assayed without themedia control, the left bar (white) represents DCs pulsed with theeducating peptide, and the right bar (striped) represents DCs pulsedwith the irrelevant peptide.

FIG. 106 shows the full-length humanized heavy chain amino acid sequenceof 2C3-SFD1 (SEQ ID NO: 272) and the full-length humanized light chainamino acid sequence of 2C3-K12 (SEQ ID NO: 273). The signal sequencesare boldfaced and italicized and the CDRs are underlined.

FIG. 107 shows the full-length humanized heavy chain amino acid sequenceof 7F11-SFD1 (SEQ ID NO: 274) and the full-length humanized light chainamino acid sequence of 7F11-K2 (SEQ ID NO: 275). The signal sequencesare boldfaced and italicized and the CDRs are underlined.

FIG. 108 shows the full-length humanized heavy chain amino acidsequences of 9D9-H16 (SEQ ID NO: 276) and 9D9-H18 (SEQ ID NO: 277), andthe full-length humanized light chain amino acid sequence of 9D9-K13(SEQ ID NO: 278). The signal sequences are boldfaced and italicized andthe CDRs are underlined.

FIG. 109 shows the full-length humanized heavy chain amino acid sequenceof 12G6-SFD1 (SEQ ID NO: 279) and the full-length humanized light chainamino acid sequence of 12G6-K12 (SEQ ID NO: 280). The signal sequencesare boldfaced and italicized and the CDRs are underlined.

FIG. 110 shows the full-length humanized heavy chain amino acid sequenceof 4B10-H1 (SEQ ID NO: 281) and the full-length humanized light chainamino acid sequence of 4B10-K1 (SEQ ID NO: 282). The signal sequencesare boldfaced and italicized and the CDRs are underlined.

FIG. 111 shows the full-length humanized heavy chain nucleic acidsequence of 2C3-SFD1 (SEQ ID NO: 283) and the full-length humanizedlight chain nucleic acid sequence of 2C3-K12 (SEQ ID NO: 284). Thesignal sequences are underlined, the variable domains are in boldface,and the constant regions are italicized.

FIG. 112 shows the full-length humanized heavy chain nucleic acidsequence of 7F11-SFD1 (SEQ ID NO: 285) and the full-length humanizedlight chain nucleic acid sequence of 7F11-K2 (SEQ ID NO: 286). Thesignal sequences are underlined, the variable domains are in boldface,and the constant regions are italicized.

FIG. 113 shows the full-length humanized heavy chain nucleic acidsequences of 9D9-H16 (SEQ ID NO: 287) and 9D9-H18 (SEQ ID NO: 288). Thesignal sequences are underlined, the variable domains are in boldface,and the constant regions are italicized.

FIG. 114 shows the full-length humanized light chain nucleic acidsequence of 9D9-K13 (SEQ ID NO: 289). The signal sequence is underlined,the variable domain is in boldface, and the constant region isitalicized.

FIG. 115 shows the full-length humanized heavy chain nucleic acidsequence of 12G6-SFD1 (SEQ ID NO: 290) and the full-length humanizedlight chain nucleic acid sequence of 12G6-K12 (SEQ ID NO: 291). Thesignal sequences are underlined, the variable domains are in boldface,and the constant regions are italicized.

FIG. 116 shows the full-length humanized heavy chain nucleic acidsequence of 4B10-H1 (SEQ ID NO: 292) and the full-length humanized lightchain nucleic acid sequence of 4B10-K1 (SEQ ID NO: 293). The signalsequences are underlined, the variable domains are in boldface, and theconstant regions are italicized.

DETAILED DESCRIPTION OF THE INVENTION

CD52 is a glycosylated, GPI anchored cell surface abundant protein(approximately 5×10⁵ antibody binding sites per cell) present on atleast 95% of all human peripheral blood lymphocytes andmonocytes/macrophages (Hale G, et al., “The CAMPATH-1 antigen (CD52),”Tissue Antigens, 35:178-327 (1990)), but is absent from hematopoieticstem cells. This invention is directed to immunoglobulins (anti-CD52)which have binding specificity (e.g., epitopic specificity) for, or areselective for binding to, human CD52 or a portion thereof. Theseimmunoglobulins bind specifically to a CD52, and do not bindspecifically to non-CD52 molecules. Specific binding between ananti-CD52 immunoglobulin and CD52 can be determined, for example, bymeasuring EC₅₀ of the immunoglobulin's binding to CD52+ cells by flowcytometry. Specific binding can be indicated by an EC₅₀ range of, e.g.,0.5-10 μg/ml. The immunoglobulins described herein can have bindingspecificity for all or a portion of a human CD52 wherein the human CD52is an isolated and/or recombinant human CD52, or on the surface of acell which expresses human CD52. In addition, the immunoglobulins canhave binding specificity for one or more forms of human CD52 (e.g.,glycosylated human CD52; de-glycosylated human CD52; non-glycosylatedhuman CD52; and allelic variants). In one embodiment, theimmunoglobulins have binding specificity for a naturally occurring,endogenous or wildtype human CD52. The amino acid sequence of a wildtypehuman CD52 is set out in FIG. 4 (SEQ ID NO: 104).

The immunoglobulins described herein can be purified or isolated usingknown techniques. Immunoglobulins that are “purified” or “isolated” havebeen separated away from molecules (e.g., peptides) of their source oforigin (e.g., the supernatant of cells; in a mixture such as in amixture of immunoglobulins in a library), and include immunoglobulinsobtained by methods described herein or other suitable methods. Isolatedimmunoglobulins include substantially pure (essentially pure)immunoglobulins, and immunoglobulins produced by chemical synthesis,recombinant techniques and a combination thereof.

More specifically, the invention relates to anti-human CD52immunoglobulins, antigen-binding fragments (i.e., portions) of theimmunoglobulins, the light chains of the immunoglobulins, the heavychains of the immunoglobulins, and fragments of these light chains orheavy chains. The invention also relates to mature immunoglobulins orchains thereof, such as glycosylated immunoglobulins. The invention alsorelates to immature or precursor immunoglobulin (protein). The inventionalso relates to nucleic acid molecules (e.g., vectors) that encode boththese immature or mature proteins, to vectors and host cells thatcomprise such nucleic acid, to methods of producing immature and matureproteins and to methods of using the immunoglobulins.

The immunoglobulins of this invention can be used to treat a subject inneed thereof (e.g., a human patient) to deplete the subject'slymphocytes and other CD52+ cells (e.g., CD52+ cancerous cells) asneeded. As used herein, “lymphocyte depletion” is a type ofimmunosuppression by reducing the population of circulating lymphocytes,e.g., T cells and/or B cells, resulting in lymphopenia. Theimmunoglobulins of this invention can also be used to inhibitangiogenesis as further described below. The immunoglobulins of thisinvention also can be used to enrich hematopoietic stem cells, forexample, in ex vivo applications (see, e.g., Lim et al., J. Hematology &Oncology 1:19 (2008)).

Naturally occurring immunoglobulins have a common core structure inwhich two identical light chains (about 24 kD) and two identical heavychains (about 55 or 70 kD) form a tetramer. The amino-terminal portionof each chain is known as the variable (V) region and can bedistinguished from the more conserved constant (C) regions of theremainder of each chain. Within the variable region of the light chain(also called the V_(L) domain) is a C-terminal portion known as the Jregion. Within the variable region of the heavy chain (also called theV_(H) domain), there is a D region in addition to the J region. Most ofthe amino acid sequence variation in immunoglobulins is confined tothree separate locations in the V regions known as hypervariable regionsor complementarity determining regions (CDRs) which are directlyinvolved in antigen binding. Proceeding from the amino-terminus, theseregions are designated CDR1, CDR2 and CDR3, respectively. The CDRs areheld in place by more conserved framework regions (FRs). Proceeding fromthe amino-terminus, these regions are designated FR1, FR2, FR3 and FR4,respectively. The locations of CDR and FR regions and a numbering systemhave been defined by Kabat et al. (Kabat, E. A., et al., Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, U.S. Government Printing Office (1991),Chothia & Lesk, Canonical Structures for the Hypervariable Regions ofImmunoglobulins, J. Mol. Biol., 196, 901-917 (1987), and the IMGT®numbering system (The International ImMunoGeneTics Iinformation System®,Lefranc, M.-P., The Immunologist 7, 132-136 (1999). Visual inspectionand sequence analysis can be carried out to identify the CDR boundaries.For this invention, the CDR sequences are defined by using both theKabat system and the IMGT system; that is, when the CDRs defined by thetwo systems do not entirely overlap, we include all the residues fromthe sequences defined by both systems.

Human immunoglobulins can be divided into classes and subclasses,depending on the isotype of the heavy chain. The classes include IgG,IgM, IgA, IgD and IgE, in which the heavy chains are of the gamma (γ),mu (μ), alpha (α), delta (δ) or epsilon (ε) type, respectively.Subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, in which theheavy chains are of the γ1, γ2, γ3, γ4, α1 and α2 type, respectively.Human immunoglobulin molecules of a selected class or subclass maycontain either a kappa (κ) or lambda (λ) light chain. See e.g., Cellularand Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50,W. B. Saunders Co., Philadelphia, Pa. 91991); Nisonoff, A., Introductionto Molecular Immunology, 2^(nd) Ed., Chapter 4, pp. 45-65, SinauerAssociates, Inc., Sunderland, Mass. (1984).

As used herein, the terms “immunoglobulin” and “antibody,” which areused interchangeably, refer to whole antibodies and antigen-bindingfragments (i.e., “antigen-binding portions”—the two terms are usedinterchangeably herein unless otherwise indicated). Antigen-bindingfragments of antibodies can be in the format of, for example, singlechain antibodies, Fv fragments, Fab fragments, Fab′ fragments, F(ab′)₂fragments, Fd fragments, single chain Fv molecules (scFv), bispecificsingle chain Fv dimers (PCT/US92/09665), diabodies, domain-deletedantibodies and single domain antibodies (dAbs). See e.g., NatureBiotechnology 22(9):1161-1165 (2004)). Also within the invention areantigen-binding molecules comprising a VH and/or a VL. In the case of aVH, the molecule may also comprise one or more of a CH1, hinge, CH2 andCH3 region. Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.

Antibody portion or fragments can be produced by enzymatic cleavage orby recombinant techniques. For instance, papain or pepsin cleavage canbe used to generate Fab or F(ab′)₂ fragments, respectively. Antibodiescan also be produced in a variety of truncated forms using antibodygenes in which one or more stop codons have been introduced upstream ofthe natural stop site. For example, a recombinant construct encoding theheavy chain of an F(ab′)₂ fragment can be designed to include DNAsequences encoding the CH₁ domain and hinge region of the heavy chain.Preferred antigen-binding fragments have binding specificity for awildtype human CD52.

In another aspect, the invention provides a variant of an antibody orportion thereof as described herein, wherein said variant binds to humanCD52 specifically but differs from the reference antibody or portionthereof by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions (forexample, in a CDR region, a FR region, or a constain domain). Forexample, the variant antibody is at least 90%, at least 91%, at least93%, at least 95%, at least 97% or at least 99% identical to thereference antibody in the heavy chain, the heavy chain variable domain,the light chain, or the light chain variable domain.

Sequence similarity or identity for polypeptides, which is also referredto as sequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000)). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially blastp or tblastn, using default parameters. See, e.g.,Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al.,Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference.

According to the invention, one type of amino acid substitution that maybe made is to change one or more cysteines in the antibody, which may bechemically reactive, to another residue, such as, without limitation,alanine or serine. In one embodiment, there is a substitution of anon-canonical cysteine. The substitution can be made in a CDR orframework region of a variable domain or in the constant domain of anantibody. In some embodiments, the cysteine is canonical. Another typeof amino acid substitution that may be made is to remove potentialproteolytic sites in the antibody. Such sites may occur in a CDR orframework region of a variable domain or in the constant domain of anantibody. Substitution of cysteine residues and removal of proteolyticsites may decrease the risk of heterogeneity in the antibody product andthus increase its homogeneity. Another type of amino acid substitutionis to eliminate asparagine-glycine pairs, which form potentialdeamidation sites, by altering one or both of the residues. In anotheraspect of the invention, the antibody may be deimmunized to reduce itsimmunogenicity using the techniques described in, e.g., PCT PublicationWO98/52976 and WO00/34317.

Another type of amino acid substitution that may be made in one of thevariants according to the invention is a conservative amino acidsubstitution. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent sequence identityor degree of similarity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well-known to those of skill in the art. See e.g.,Pearson, Methods Mol. Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartic acid and glutamic acid; and 7)sulfur-containing side chains: cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992). A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

In certain embodiments, amino acid substitutions to an antibody orantigen-binding portion of the invention are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, for example,to enhance ADCC and CDC activity of the antibody, (4) confer or modifyother physicochemical or functional properties of such analogs, butstill retain specific binding to human CD52, (5) remove C-terminallysine, and (6) add or remove glycosylation sites.

In an aspect, the invention provides a new and novel polypeptide that isthe heavy or light chain of an antibody of this invention, or that is avariable domain-containing portion of the heavy or light chain. Such apolypeptide is useful because it can partner with an opposite (light orheavy) antibody chain to form a CD52-binding molecule.

Humanized Immunoglobulins

Described herein are humanized immunoglobulins comprising the CDRs ofnovel mouse anti-human CD52 antibodies. In one embodiment, the humanizedimmunoglobulin comprises a humanized light chain and a humanized heavychain that have CDR amino acid sequences which differ from the aminoacid sequence of other humanized versions of anti-CD52 antibodies (e.g.,Campath®).

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising chains that comprise one or more light chainCDRs (CDR1, CDR2 and CDR3) and one or more heavy chain CDRs (CDR1, CDR2and CDR3) of an anti-CD52 antibody of non-human origin, also referred toherein as the donor antibody (e.g., a murine anti-CD52 antibody), and atleast a portion of an immunoglobulin of human origin (e.g., frameworkregions, or framework and constant regions, derived from a light and/orheavy chain of human origin, such as CDR-grafted antibodies with orwithout framework changes). The humanized immunoglobulin of theinvention comprises at least one CDR that differs from at least one CDR(e.g., from the corresponding CDR) present in Campath®. See, e.g.,Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E.A. et al., European Patent Application No. 0,519,596 A1. See also,Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786;and Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding singlechain antibodies. In some embodiments, humanized immunoglobulins arede-immunized antibodies. See, e.g., Carr et al., U.S. Pat. No.7,264,806, regarding de-immunized immunoglobulins that have beenmodified to reduce the number of potential T-cell epitopes, therebyreducing the propensity for the immunoglobulin to elicit an immuneresponse upon administration to a human.

In particular embodiments, the humanized immunoglobulin comprises one ormore light chain CDRs and one or more heavy chain CDRs of one or more ofthe following murine monoclonal antibodies: mouse 8G3.25.3.5, mouse4G7.F3, mouse 9D9.A2, mouse 11C11.C5, mouse 3G7.E9, mouse 5F7.1.1.4,mouse 12G6.15.1.2, mouse 23E6.2.2.1, mouse 2C3.3.8.1, mouse 7F11.1.9.7,and mouse 4B10.1.2.4.

In another embodiment, the humanized immunoglobulins bind human CD52with an affinity similar to or better than that of Campath®. In aparticular embodiment, the humanized immunoglobulin of the presentinvention has the binding specificity of a murine anti-human CD52antibody of the invention (e.g., having specificity for human CD52,having the same or similar epitopic specificity) and/or it has the sameinhibitory function. The humanized immunoglobulins can have the bindingspecificity and/or inhibitory activity of a murine anti-human CD52antibody or humanized anti-human CD52 antibody described herein, and/orthe epitopic specificity of a murine anti-human CD52 antibody orhumanized anti-human CD52 antibody described herein (e.g., it cancompete with the murine anti-human CD52 antibody, or another humanizedanti-CD52 antibody (e.g., Campath®) for binding to CD52, and/or it canhave the inhibitory function of the murine or humanized anti-human CD52antibody). In a particular embodiment, the humanized immunoglobulin hasthe binding specificity, epitopic specificity and/or inhibitory activityof any one of mouse antibodies 8G3, 4G7, 9D9, 11C11, 3G7, 5F7, 12G6,23E6, 2C3, 7F11, andr 4B10.

The portion of the humanized immunoglobulin or immunoglobulin chainwhich is of human origin (e.g., framework region; constant region) canbe derived from any suitable human immunoglobulin or immunoglobulinchain. For example, a human constant region or portion thereof in ahumanized or chimeric antibody can be derived from a human κ or λ lightchain gene, and/or from a human γ (e.g., γ1, γ2, γ3, γ4), μ, α (e.g.,α1, α2), δ or ε heavy chain gene, including allelic variants. Aparticular constant region (e.g., IgG1), variant or portion thereof canbe selected in order to tailor effector function. For example, a mutatedconstant region (variant) can be incorporated into the immunoglobulin orimmunoglobulin chain so as to minimize binding to Fc receptors and/orability to fix complement. (See e.g., Winter et al., GB 2,209,757 B;Morrison et al., WO 89/07142; Morgan et al., WO 94/29351, Dec. 22,1994). In one embodiment, the human framework has no variation ormutation in its structure or sequence. In a particular embodiment, theframework is a germline framework sequence that has no mutations orvariations in its sequence.

As used herein, the term “germline” refers to the nucleotide sequencesand amino acid sequences of the antibody genes and gene segments as theyare passed from parents to offspring via the germ cells. This germlinesequence is distinguished from the nucleotide sequences encodingantibodies in mature B cells which have been altered by recombinationand hypermutation events during the course of B cell maturation. Anantibody that “utilizes” a particular germline has a nucleotide or aminoacid sequence that most closely aligns with that germline nucleotidesequence or with the amino acid sequence that it specifies. Suchantibodies frequently are mutated compared with the germline sequence.

In other embodiments, the human framework has minimal variation ormutation from germline sequence in its structure or sequence (e.g., lessthan 3, 4, 5, 6, 7, 8, 9, or 10 acceptor framework residues have beenreplaced with donor framework residues to improve binding affinity, seeQueen et al., U.S. Pat. No. 5,530,101). In a particular embodiment, alimited number of amino acids in the framework of a humanizedimmunoglobulin chain (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids) are chosen to be the same as the amino acids at those positionsin the donor sequence (i.e., “back-mutated”), rather than in theacceptor sequence, to increase the affinity of an antibody comprisingthe humanized immunoglobulin chain for human CD52.

Human framework regions (e.g., of the heavy and/or light chain variableregions) are preferably obtained or derived from a human antibodyvariable region having sequence similarity to the analogous orequivalent region (e.g., heavy or light chain variable regions) of theantigen-binding region of the donor immunoglobulin (murine anti-CD52antibody). Other sources of framework regions for portions of humanorigin of a humanized immunoglobulin include human variable regionconsensus sequences (See e.g., Kettleborough, C. A. et al., ProteinEngineering 4:773-783 (1991); Carter et al., WO 94/04679; Carter U.S.Pat. No. 6,407,213)). For example, the region of the donor sequence ofthe antibody (e.g., the sequence of the variable region) used to obtainthe nonhuman portion can be compared to human sequences as described inKabat, E. A. et al. Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, U.S.Government Printing Office (1991) to select a particular source of thehuman portions of the humanized immunoglobulin, e.g., a source of theframework regions.

In one embodiment, the framework regions of the humanized immunoglobulinchains are obtained, or derived, from a human Ig variable region havingat least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% overallsequence identity, with the variable region of the nonhuman donor. In aparticular embodiment, the framework regions of the humanizedimmunoglobulin chains are obtained or derived from human variable regionframework regions having at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, or atleast about 95% overall sequence identity, with the framework regions ofthe variable region of the nonhuman donor immunoglobulin.

In one embodiment, at least one of the framework regions (FR) of thehumanized immunoglobulin is obtained or derived from one or more chainsof an antibody of human origin. Thus, the FR can include a FR1 and/orFR2 and/or FR3 and/or FR4 obtained or derived from one or moreantibodies of human origin (e.g., from a human immunoglobulin chain,from a human consensus sequence).

The immunoglobulin portions for use in the present invention havesequences identical, or similar, to immunoglobulins from which they arederived or to variants thereof. Such variants include mutants differingby the addition, deletion or substitution (e.g., conservativesubstitution) of one or more residues, e.g., differing by up to 3, 4, 5,6, 7, 8, 9, or 10 residues from the parental sequence by one or moreadditions, deletions or substitutions. As indicated above, the humanizedimmunoglobulin of the invention comprises one or more CDRs from one ormore of the murine anti-CD52 antibodies (donor antibodies) describedherein. Changes in the framework region, such as those which substitutea residue of the framework region of human origin with a residue fromthe corresponding position of the donor antibody, can be made. One ormore mutations, including deletions, insertions and substitutions of oneor more amino acids in the framework region, can be made. If desired,framework mutations can be included in a humanized antibody or chain,and sites for mutation can be selected using any suitable method, forexample as described in WO 98/06248, the entire teachings of which areincorporated by reference.

It will be appreciated by one of skill in the art that in some casesresidues flanking the one or more CDRs of the murine anti-CD52antibody(ies) may contribute, and in some cases, may be essential,either directly or indirectly, to function (e.g., binding). Thus, insome embodiments, one or more amino acids flanking one or more CDRs(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 flanking amino acids) of the murineframework are also included in the humanized immunoglobulin.

In some embodiments, the human heavy chain framework regions of thehumanized antibodies of this invention utilize the human VH3-72 orVH3-23 germline sequence. In some embodiments, the human light chainframework regions of the humanized antibodies of this invention utilizethe human Vk2-A18b germline sequence. Back mutations may optionally bemade in these FR regions at one or more of the residues as described inthe Working Examples below to improve CD52-binding affinity of thehumanized antibody.

“Affinity” is a term of art that describes the strength of a bindinginteraction and typically refers to the overall strength of binding ofthe immunoglobulin to human CD52.

In a particular embodiment, the immunoglobulin has a binding activitymeasured as an EC₅₀ value of less than 10 μg/ml (e.g., as determined byflow cytometry). In another embodiment, the immunoglobulin has a bindingactivity measured as an EC₅₀ value of less than 5.0 μg/ml, or less than1.0 μg/ml (e.g., as determined by flow cytometry).

In some embodiments, the immunoglobulin binds to human CD52 with anaffinity (K_(D); K_(D)=K_(off)(kd)/Kon (ka)) of 300 nM to 1 pM (i.e.,3×10⁻⁷ to 1×10⁻¹²M), preferably 50 nM to 1 pM, more preferably 5 nM to 1pM and most preferably 1 nM to 1 pM, for example, a K_(D) of 1×10⁻⁷ M orless, preferably 1×10⁻⁸ M or less, more preferably 1×10⁻⁹ M or less,advantageously 1×10⁻¹⁰ M or less and most preferably 1×10⁻¹¹ M or1××10⁻¹² or less; and/or a K_(off) rate constant of 5×10⁻¹ s−1 to 1×10⁻⁷s−1, preferably 1×10⁻² s−1 to 1×10⁻⁶ s−1, more preferably 5×10⁻³ s−1 to1×10⁻⁵ s−1, for example 5×10⁻¹ s−1 or less, preferably 1×10⁻² s−1 orless, advantageously 1×10⁻³ s−1 or less, more preferably 1×10⁴ s−1 orless, still more preferably 1×10⁻⁵ s−1 or less, and most preferably1×10⁻⁶ s−1 or less as determined by surface plasmon resonance.

As is apparent to one of skill in the art, a variety of methods can beused to confirm that immunoglobulins produced according to methodsprovided herein and known in the art have the requisite specificity(e.g., binding specificity, epitopic specificity). For example, thebinding function of a humanized anti-CD52 immunoglobulin of theinvention having binding specificity for human CD52 can be detectedusing any suitable method, e.g., assays which monitor formation of acomplex between humanized immunoglobulin and human CD52 (e.g., amembrane fraction comprising human CD52; a cell bearing human CD52, suchas a human T cell, a human B cell; a CHO cell or a recombinant host cellcomprising and expressing a nucleic acid encoding human CD52; a peptide(e.g., a synthetic peptide) having an amino acid sequence of CD52; asolid support comprising human CD52).

The ability of an immunoglobulin of the invention (e.g., a humanizedimmunoglobulin of the invention) to bind to the same epitope on humanCD52 as a particular murine, chimeric, or humanized monoclonal antibody,or to bind to an epitope on human CD52 which overlaps with the epitopeon human CD52 to which a particular murine, chimeric, or humanizedmonoclonal antibody binds, can be readily determined using a variety oftechniques known to those of skill in the art, including e.g.,competitive binding assays. These may involve the use of a labeled formof said particular antibody, and a measurement of the binding of thatlabeled antibody to human CD52 in the presence and in the absence of animmunoglobulin of the invention.

An “epitope” as used herein includes any protein determinant capable ofspecific binding to an immunoglobulin. Epitopic determinants generallyconsist of chemically active surface groupings of molecules such asamino acids or carbohydrate or sugar side chains and generally havespecific three dimensional structural characteristics, as well asspecific charge characteristics. An epitope may be “linear” or“conformational.” In a linear epitope, all of the points of interactionbetween the protein and the interacting molecule (such as an antibody)occur linearly along the primary amino acid sequence of the protein. Ina conformational epitope, the points of interaction occur across aminoacid residues on the protein that are separated from one another. Once adesired epitope on an antigen is determined, it is possible to generateantibodies to that epitope, e.g., using the techniques described in thepresent invention. Alternatively, during the discovery process, thegeneration and characterization of antibodies may elucidate informationabout desirable epitopes. From this information, it is then possible tocompetitively screen antibodies for binding to the same epitope. Anapproach to achieve this is to conduct competition studies to findantibodies that competitively bind with one another, i.e., theantibodies compete for binding to the antigen.

In one embodiment, to determine if a test antibody binds to the same oroverlapping epitope of a humanized antibody of this invention, oneallows the anti-CD52 antibody of the invention to bind to CD52 undersaturating conditions and then measures the ability of the test antibodyto bind to CD52. If the test antibody is able to bind to CD52 at thesame time as the reference anti-CD52 antibody, then the test antibodybinds to a different epitope than the reference anti-CD52 antibody.However, if the test antibody is not able to bind to CD52 at the sametime, then the test antibody binds to the same epitope, an overlappingepitope, or an epitope that is in close proximity to the epitope boundby the anti-CD52 antibody of the invention. This experiment can beperformed using ELISA, RIA, BIACORE™, or flow cytometry. To test whetheran anti-CD52 antibody cross-competes with another anti-CD52 antibody,one may use the competition method described above in two directions,i.e., determining if the reference antibody blocks the test antibody andvice versa. In a some embodiment, the experiment is performed usingBIACORE™.

Epitope binning can also be useful to characterize the antibodies ofthis invention. The term “binning” refers to a method to groupantibodies based on their antigen binding characteristics. A highthroughput process for “binning” antibodies based upon theircross-competition is described in International Patent Application No.WO 03/48731. The “epitope binning” can be investigated by allowing anunlabeled form of an anti-CD52 antibody “A” to bind to a syntheticpeptide corresponding to the sequence of CD52 or to CD52 positive cells.Subsequently a labeled second anti-CD52 antibody “B” is added and onecan assess the amount of labeled antibody that can bind relative to acontrol sample where the cells or synthetic peptide have not beenexposed previously to anti-CD52 antibody “A.” Alternatively, anti-CD52antibodies “A” and “B” can both be labeled with different fluorochromesor chemicals enabling detection, and one can measure the quantities ofboth labeled antibodies that can engage the CD52 peptide at the sametime using a device capable of detecting the label or measure theamounts of both antibodies that simultaneously engage CD52 positivecells by flow cytometry. Biacore and Octet technologies enable one toinvestigate the competitive binding of unlabelled forms of antibodies.This use of unlabelled forms of antibodies is desired as the chemicalmodification of some antibodies can compromise the binding activity. Seealso the technology described in See also Jia et al., J. Immunol.Methods 288:91-98 (2004), which is useful in performing epitope binningas well.

Also provided herein are portions of the humanized immunoglobulins suchas light chains, heavy chains and portions of light and heavy chains.These immunoglobulin portions can be obtained or derived fromimmunoglobulins (e.g., by reduction and/or cleavage), or produced orexpressed by nucleic acids encoding a portion of an immunoglobulin orchain thereof having the desired property (e.g., binds human CD52,sequence similarity). They can be prepared by e.g., de novo synthesis ofthe relevant portion. Humanized immunoglobulins comprising the desiredportions (e.g., antigen-binding region, CDR, FR, C region) of human andnonhuman origin can be produced using synthetic and/or recombinantnucleic acids to prepare constructs (e.g., cDNA) encoding the desiredhumanized chain. For example, to prepare a portion of an immunoglobulin(e.g., a portion of a chain), one or more stop codons can be introducedat the desired position. Nucleic acid (e.g., DNA) sequences coding forhumanized variable regions can be constructed using PCR mutagenesismethods to alter existing DNA sequences (see e.g., Kamman, M., et al.,Nucl. Acids Res. 17:5404 (1989)). PCR primers coding for the new CDRscan be hybridized to a DNA template of a previously humanized variableregion which is based on the same, or a very similar, human variableregion (Sato, K., et al., Cancer Research 53:851-856 (1993)). If asimilar DNA sequence is not available for use as a template, a nucleicacid comprising a sequence encoding a variable region sequence can beconstructed from synthetic oligonucleotides (see e.g., Kolbinger, F.,Protein Engineering 8:971-980 (1993)). A sequence encoding a signalpeptide can also be incorporated into the nucleic acid (e.g., onsynthesis, upon insertion into a vector). If a signal peptide sequenceis unavailable (e.g., not typically present), a signal peptide sequencefrom another antibody can be used (see, e.g., Kettleborough, C. A.,Protein Engineering 4:773-783 (1991)). Using these methods, methodsdescribed herein or other suitable methods, variants can readily beproduced.

The invention relates to a humanized immunoglobulin that has bindingspecificity for human CD52 and comprises a humanized light chain and ahumanized heavy chain and/or portions thereof. In one embodiment, thehumanized immunoglobulin comprises a light chain comprising one or moreCDRs (e.g., all three CDRs) of SEQ ID NO: 3 and a heavy chain comprisingone or more CDRs (e.g., all three CDRs) of SEQ ID NO: 16; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 4 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 17; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 5 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 18; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 6 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 19; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 7 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 20; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 8 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 21; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 9 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 22; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 10 anda heavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQID NO: 23; a light chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 11 and a heavy chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 24; a light chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 12 and a heavy chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 25; ora light chain comprising one or more CDRs (e.g., all three CDRs) of SEQID NO: 13 and a heavy chain sequence comprising one or more CDRs (e.g.,all three CDRs) of SEQ ID NO: 26.

In one embodiment, a humanized immunoglobulin of the invention comprisesheavy chain (H)-CDR1, H-CDR2, H-CDR3, light chain (L)-CDR1, L-CDR2, andL-CDR3 whose amino acid sequences are: a) SEQ ID NOs: 51, 59, 69, 29,36, and 43, respectively; b) SEQ ID NOs: 50, 60, 69, 29, 37, and 43,respectively; c) SEQ ID NOs: 50, 61, 68, 29, 38, and 43, respectively;d) SEQ ID NOs: 50, 61, 69, 29, 36, and 43, respectively; e) SEQ ID NOs:50, 62, 69, 29, 39, and 43, respectively; f) SEQ ID NOs: 52, 61, 70, 30,40, and 43, respectively; g) SEQ ID NOs: 53, 63, 71, 31, 36, and 44,respectively; h) SEQ ID NOs: 54, 64, 71, 31, 36, and 45, respectively;i) SEQ ID NOs: 55, 63, 72, 31, 36, and 46, respectively; j) SEQ ID NOs:56, 65, 73, 32, 41, and 47, respectively; k) SEQ ID NOs: 56, 65, 294,32, 41, and 47; or l) SEQ ID NOs: 56, 66, 74, 33, 41, and 48,respectively.

In another embodiment, a humanized immunoglobulin of this inventioncomprises H-CDR3 and L-CDR3 whose sequences are a) SEQ ID NOs: 69 and43, respectively; b) SEQ ID NOs: 68 and 43, respectively; c) SEQ ID NOs:70 and 43, respectively; d) SEQ ID NOs: 71 and 44, respectively; e) SEQID NOs: 71 and 45, respectively; f) SEQ ID NOs: 72 and 46, respectively;g) SEQ ID NOs: 73 and 47, respectively; h) SEQ ID NOs: 294 and 47,respectively; or i) SEQ ID NOs: 74 and 48, respectively.

In another embodiment, the humanized immunoglobulin has bindingspecificity for human CD52 and comprises a light chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ IDNO: 47, and SEQ ID NO: 48, or a combination thereof; and a heavy chaincomprising one or more CDRs selected from the group consisting of SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ IDNO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 294, or a combination thereof,wherein the humanized immunoglobulin is not Campath®.

In another embodiment, the humanized immunoglobulin that has a bindingspecificity for human CD52 comprises a light chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, and a heavy chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, or SEQ ID NO: 137, wherein the humanized immunoglobulin is notCampath®.

The invention also relates to a humanized immunoglobulin light chain ofthe humanized immunoglobulin described herein. In one embodiment, thehumanized immunoglobulin light chain comprises one or more CDRs selectedfrom the group consisting of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ IDNO: 48, and a combination thereof, wherein the humanized immunoglobulinlight chain is not the light chain of Campath®. For example, thehumanized antibody has L-CDR1, L-CDR2, and L-CDR3 whose amino acidsequences are: a) SEQ ID NOs: 29, 36, and 43, respectively; b) SEQ IDNOs: 29, 37, and 43, respectively; c) SEQ ID NOs: 29, 38, and 43,respectively; d) SEQ ID NOs: 29, 36, and 43, respectively; e) SEQ IDNOs: 29, 39, and 43, respectively; f) SEQ ID NOs: 30, 40, and 43,respectively; g) SEQ ID NOs: 31, 36, and 44, respectively; h) SEQ IDNOs: 31, 36, and 45, respectively; i) SEQ ID NOs: 31, 36, and 46,respectively; j) SEQ ID NOs: 32, 41, and 47, respectively; or k) SEQ IDNOs: 33, 41, and 48, respectively.

The invention also relates to humanized heavy chain comprising one ormore CDRs selected from the group consisting of SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 74, and SEQ ID NO: 294, or a combination thereof, wherein thehumanized immunoglobulin heavy chain is not the heavy chain of Campath®.For example, the humanized antibody has H-CDR1, H-CDR2, and H-CDR3 whoseamino acid sequences are: a) SEQ ID NOs: 51, 59, and 69, respectively;b) SEQ ID NOs: 50, 60, and 69, respectively; c) SEQ ID NOs: 50, 61, and68, respectively; d) SEQ ID NOs: 50, 61, and 69, respectively; e) SEQ IDNOs: 50, 62, and 69, respectively; f) SEQ ID NOs: 52, 61, and 70,respectively; g) SEQ ID NOs: 53, 63, and 71, respectively; h) SEQ IDNOs: 54, 64, and 71, respectively; i) SEQ ID NOs: 55, 63, and 72,respectively; j) SEQ ID NOs: 56, 65, and 73, respectively; k) SEQ IDNOs: 56, 65, and 294; or l) SEQ ID NOs: 56, 66, and 74, respectively.

In one embodiment, a humanized antibody of this invention comprises alight chain comprising a variable domain (V_(L)) sequence of one of SEQID NOs: 102, 138, 145-148, 153-157, and 164-168. In a relatedembodiment, the humanized antibody comprises a light chain whose aminoacid sequence comprises or consists of one of SEQ ID NOs: 273, 275, 278,280, and 282.

In one embodiment, a humanized antibody of this invention comprises aheavy chain comprising a variable domain (V_(H)) sequence of one of SEQID NOs: 103, 136, 137, 139-144, 149-152, and 158-163. In a relatedembodiment, the humanized antibody comprises a heavy chain whose aminoacid sequence comprises or consists of one of SEQ ID NOs: 272, 274, 276,277, 279, and 281.

In some embodiments, a humanized antibody of this invention comprises aV_(H) and a V_(L) whose amino acid sequences comprise or consist of

a) SEQ ID NOs: 103 and 102, respectively (4B10-H1/K1);

b) SEQ ID NOs: 136 and 138, respectively (7F11-SFD1/K2);

c) SEQ ID NOs: 137 and 138, respectively (7F11-SFD2/K2)

d) SEQ ID NO: 139 and one of SEQ ID NOs: 145-148, respectively (e.g.,SEQ ID NOs: 139 and 146, respectively (2C3-SFD1/K11); and SEQ ID NOs:139 and 147, respectively (2C3-SFD1/K12));

e) SEQ ID NO: 140 and one of SEQ ID NOs: 145-148, respectively;

f) SEQ ID NO: 141 and one of SEQ ID NOs: 145-148, respectively;

g) SEQ ID NO: 142 and one of SEQ ID NOs: 145-148, respectively;

h) SEQ ID NO: 143 and one of SEQ ID NOs: 145-148, respectively;

i) SEQ ID NO: 144 and one of SEQ ID NOs: 145-148, respectively;

j) SEQ ID NO: 149 and one of SEQ ID NOs: 153-157, respectively (e.g.,SEQ ID NOs: 149 and 155, respectively (12G6-SFD1/K11); SEQ ID NOs: 149and 156, respectively (12G6-SFD1/K12); and SEQ ID NOs: 149 and 157,respectively (12G6-SFD1/K13));

k) SEQ ID NO: 150 and one of SEQ ID NOs: 153-157, respectively;

l) SEQ ID NO: 151 and one of SEQ ID NOs: 153-157, respectively;

m) SEQ ID NO: 152 and one of SEQ ID NOs: 153-157, respectively;

n) SEQ ID NO: 158 and one of SEQ ID NOs: 164-168, respectively (e.g.,SEQ ID NOs: 158 and 165, respectively (9D9-H10/K12); and SEQ ID NOs: 158and 166, respectively (9D9-H10/K13));

o) SEQ ID NO: 159 and one of SEQ ID NOs: 164-168, respectively (e.g.,SEQ ID NOs: 159 and 165, respectively (9D9-H11/K12); and SEQ ID NOs: 159and 166, respectively (9D9-H11/K13));

p) SEQ ID NO: 160 and one of SEQ ID NOs: 164-168, respectively;

q) SEQ ID NO: 161 and one of SEQ ID NOs: 164-168, respectively (e.g.,SEQ ID NOs: 161 and 166, respectively (9D9-H16/K13));

r) SEQ ID NO: 162 and one of SEQ ID NOs: 164-168, respectively; or

s) SEQ ID NO: 163 and one of SEQ ID NOs: 164-168, respectively (e.g.,SEQ ID NOs: 163 and 166, respectively (9D9-H18/K13)).

The antibodies included in the parentheses are further described belowin the working examples.

In one embodiment, a humanized antibody of this invention comprises alight chain (LC) and a heavy chain (HC) whose amino acid sequencescomprise or consist of a) SEQ ID NOs: 273 and 272, respectively; b) SEQID NOs: 275 and 274, respectively; c) SEQ ID NOs: 278 and 276,respectively; d) SEQ ID NOs: 278 and 277, respectively; e) SEQ ID NOs:280 and 279, respectively; or f) SEQ ID NOs: 282 and 281, respectively.

This invention also provides anti-human CD52 antibodies (except those,in any, known in the prior art) that binds to the same epitope as, orcompetes or cross-competes with, an antibody exemplified herein. Theseantibodies can be, for example, humanized, chimeric, or mouseantibodies. For example, the invention provides anti-human CD52antibodies that bind to the same epitope as, or competes orcross-competes with, one of mouse antibodies 8G3, 4F7, 9D9, 11C11, 3G7,5F7, 12G6, 23E6, 2C3, 7F11, and 4B10, and humanized and chimericversions of these mouse antibodies. The ability of an antibody to bindto the same epitope as, or competes or cross-competes with a referenceantibody can be determined as described above. For example, we havefound that the CD52 epitope bound by the humanized antibodies2C3-SFD1/K12 and 12G6-SFD1/K12 includes residues 7, 8, and 11 in SEQ IDNO: 104, and that the epitope bound by the humanized antibody9D9-H16/K13 includes residues 4 and 11 in SEQ ID NO: 104. Thus, in someembodiments, this invention provides anti-CD52 antibodies that bind tothe same epitope as, or competes or cross-competes with, those humanizedantibodies.

If desired, for example, for diagnostic or assay purposes (e.g., imagingto allow, for example, monitoring of therapies), the humanizedimmunoglobulin (e.g., antigen-binding fragment thereof) can comprise adetectable label. Suitable detectable labels and methods for labeling ahumanized immunoglobulin or antigen-binding fragment thereof are wellknown in the art. Suitable detectable labels include, for example, aradioisotope (e.g., as Indium-111, Technetium-99m or Iodine-131),positron emitting labels (e.g., Fluorine-19), paramagnetic ions (e.g.,Gadlinium (III), Manganese (II)), an epitope label (tag), an affinitylabel (e.g., biotin, avidin), a spin label, an enzyme, a fluorescentgroup or a chemiluminescent group. When labels are not employed, complexformation (e.g., between humanized immunoglobulin and human CD52) can bedetermined by surface plasmon resonance, ELISA, FACS, or other suitablemethods.

Anti-CD52 antibodies used in the invention also may be conjugated, via,for example, chemical reactions or genetic modifications, to othermoieties (e.g., pegylation moieties) that improve the antibodies'pharmacokinetics such as half-life. In some embodiments, the anti-CD52antibodies used in this invention can be linked to a suitable cytokinevia, e.g., chemical conjugation or genetic modifications (e.g.,appending the coding sequence of the cytokine in frame to an antibodycoding sequence, thereby creating an antibody:cytokine fusion protein).

The invention also relates to immunoconjugates in which the humanizedimmunoglobulin (e.g., antigen-binding fragment thereof) of the inventionis coupled to another therapeutic agent, such as a bioactive compound(e.g., cytokines, superantigens, cytotoxic agents and toxins). Forexample, the humanized immunoglobulin that has binding specificity forhuman CD52 (e.g., antigen binding fragment thereof) can be coupled to abiological protein, a molecule of plant or bacterial origin (orderivative thereof), an interleukin-2 antibody or diptheria toxinantibodies.

Mouse Monoclonal Immunoglobulins

As described herein, mouse monoclonal immunoglobulins having bindingspecificity for human CD52 have been produced Humanized and chimericantibodies of this invention can be derived from the mouse monoclonalantibodies of this invention. That is, in some embodiments, humanizedand chimeric anti-CD52 antibodies of the invention comprise sequencestaken from a mouse monoclonal antibody of the invention, such as one ormore CDR sequences. A mouse monoclonal immunoglobulin of this inventioncomprises a light chain and a heavy chain that have CDR amino acidsequences which differ from the CDR amino acid sequences of known mouseanti-CD52 monoclonal antibodies (e.g., from CF1D12).

As used herein, the term “mouse monoclonal immunoglobulin” refers to animmunoglobulin containing light chain CDRs (L-CDR1, L-CDR2 and L-CDR3)and heavy chain CDRs (H-CDR1, H-CDR2 and H-CDR3) of a murine anti-humanCD52 antibody, and framework and constant regions of murine origin.Mouse monoclonal immunoglobulins are homogeneous antibodies of a singlespecificity prepared, for example, by the use of hybridoma technology orrecombinant methods.

The invention relates to the mouse monoclonal immunoglobulins describedherein, including antigen-binding fragments (i.e., portions) of themouse monoclonal immunoglobulins, the light chains of the mousemonoclonal immunoglobulins, the heavy chains of the mouse monoclonalimmunoglobulins, and fragments of these heavy and light chains. In aparticular embodiment, the mouse monoclonal antibody is the mouse8G3.25.3.5 (also called GENZ 8G3.25.3.5 or 8G3), mouse GMA 4G7.F3 (alsocalled 4G7.F3 or 4G7), mouse GMA 9D9.A2 (also called 9D9.A2 or 9D9),mouse GMA 11C11.C5 (also called 11C11.C5 or 11C11), mouse GMA 3G7.E9(also called 3G7.E9 or 3G7), mouse 5F7.1.1.4 (also called GENZ 5F7.1.1.4or 5F7), mouse 12G6.15.1.2 (also called GENZ 12G6.15.1.2 or 2G6), mouse23E6.2.2.1 (also called GENZ 23E6.2.2.1 or 23E6), mouse 2C3.3.8.1 (alsocalled GENZ 2C3.3.8.1 or 2C3), mouse 7F11.1.9.7 (also called GENZ7F11.1.9.7 or 7F11), or mouse 4B10.1.2.4 (also called GENZ 4B10.1.2.4 or4B10). The invention relates to mature mouse monoclonal immunoglobulin,such as the mouse monoclonal immunoglobulin following processing toremove the heavy and light chain signal peptides and/or to theglycosylated immunoglobulin. The invention also relates to immature orprecursor protein, such as a mouse immunoglobulin light chain or a mouseimmunoglobulin heavy chain comprising a signal peptide. The inventionalso relates to nucleic acid molecules (e.g., vectors) that encode theseimmature or mature proteins, to host cells that comprise such nucleicacids and to methods of producing these immature and mature proteins.

The binding function of a mouse monoclonal immunoglobulin having bindingspecificity for human CD52 can be detected using any suitable method,for example using assays which monitor formation of a complex betweenmouse monoclonal immunoglobulin and human CD52 (e.g., a membranefraction comprising human CD52, or a cell bearing human CD52, such as ahuman T cell, a human B cell, CHO cell or a recombinant host cellcomprising a nucleic acid encoding human CD52; a peptide (e.g., asynthetic peptide) having an amino acid sequence of CD52).

Also provided herein are portions of the murine immunoglobulins whichinclude light chains, heavy chains and portions of light and heavychains. These immunoglobulin portions can be obtained or derived fromimmunoglobulins (e.g., by reduction and/or cleavage), or nucleic acidsencoding a portion of an immunoglobulin or chain thereof having thedesired property (e.g., binds human CD52, sequence similarity) can beproduced and expressed. They can be prepared by e.g., de novo synthesisof a portion of mouse monoclonal immunoglobulins comprising the desiredportions (e.g., antigen-binding region, CDR, FR, and/or C region) ofmurine origin can be produced using synthetic and/or recombinant nucleicacids to prepare constructs (e.g., cDNA) encoding the desired monoclonalimmunoglobulin chain. To prepare a portion of a chain, one or more stopcodons can be introduced at the desired position. A sequence encoding asignal peptide can also be incorporated into the nucleic acid (e.g., onsynthesis, upon insertion into a vector). If the natural signal peptidesequence is unavailable, a signal peptide sequence from another antibodycan be used (see, e.g., Kettleborough, C. A., Protein Engineering4:773-783 (1991)). Using these methods, methods described herein orother suitable methods, variants can be readily produced.

In one embodiment, a mouse monoclonal immunoglobulin of this inventioncomprises a light chain comprising SEQ ID NO: 3 and a heavy chaincomprising SEQ ID NO: 16; a light chain comprising SEQ ID NO: 4 and aheavy chain comprising SEQ ID NO: 17; a light chain comprising SEQ IDNO: 5 and a heavy chain comprising SEQ ID NO: 18; a light chaincomprising SEQ ID NO: 6 and a heavy chain comprising SEQ ID NO: 19; alight chain comprising SEQ ID NO: 7 and a heavy chain comprising SEQ IDNO: 20; a light chain comprising SEQ ID NO: 8 and a heavy chaincomprising SEQ ID NO: 21; a light chain comprising SEQ ID NO: 9 and aheavy chain comprising SEQ ID NO: 22; a light chain comprising SEQ IDNO: 10 and a heavy chain comprising SEQ ID NO: 23; a light chaincomprising SEQ ID NO: 11 and a heavy chain comprising SEQ ID NO: 24; alight chain comprising SEQ ID NO: 12 and a heavy chain comprising SEQ IDNO: 25; or a light chain comprising SEQ ID NO: 13 and a heavy chaincomprising SEQ ID NO: 26.

In another embodiment, the invention also relates to a mouse monoclonalantibody that has binding specificity for human CD52, comprising a lightchain variable region selected from the group consisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11, SEQ ID NO: 12, and SEQ IDNO: 13; and a heavy chain variable region selected from the groupconsisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23; SEQ IDNO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

If desired, for example, for diagnostic or assay purposes (e.g.,imaging), the mouse monoclonal immunoglobulin (e.g., antigen bindingfragment thereof) can comprise a detectable label. Suitable detectablelabels and methods for labeling a mouse monoclonal immunoglobulin arewell known in the art. Suitable detectable labels include, for example,a radioisotope (e.g., as Indium-111, Technnetium-99m or Iodine-131),positron emitting labels (e.g., Fluorine-19), paramagnetic ions (e.g.,Gadlinium (III), Manganese (II)), an epitope label (tag), an affinitylabel (e.g., biotin, avidin), a spin label, an enzyme, a fluorescentgroup or a chemiluminescent group. When labels are not employed, complexformation (e.g., between mouse monoclonal immunoglobulin and CD52) canbe determined by surface plasmon resonance or other suitable methods.All suitable methods and techniques described above for humanizedantibodies of this invention can also be used herein.

Chimeric Immunoglobulins

As described herein, chimeric immunoglobulins having binding specificityfor human CD52 have been produced. The chimeric immunoglobulin comprisesa chimeric light chain and/or a chimeric heavy chain that have aminoacid sequences which differ from the amino acid sequence of knownchimeric antibodies having binding specificity for human CD52.

As used herein, the term “chimeric immunoglobulin” refers to arecombinant protein that contains the variable domains including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a murine anti-human CD52 monoclonal antibody,while the constant domains of the antibody molecule are derived fromthose of a different species, e.g., from a human antibody.

The invention relates to the chimeric immunoglobulins described herein,including antigen-binding fragments (i.e., portions) of the chimericimmunoglobulins, the chimeric light chains and chimeric heavy chains ofthe chimeric immunoglobulins and fragments of these chimeric light andheavy chains. The invention relates to mature chimeric immunoglobulin,such as the chimeric immunoglobulin following processing to remove theheavy and light signal peptides and/or to the glycosylatedimmunoglobulin. The invention also relates to immature or precursorprotein, such as a chimeric heavy chain comprising a signal peptide. Theinvention also relates to nucleic acid molecules (e.g., vectors) thatencode these immature or mature proteins, to host cells that comprisesuch nucleic acids and to methods of producing these immature and matureproteins.

The binding function of a chimeric immunoglobulin having bindingspecificity for human CD52 can be detected using any suitable method,for example using assays which monitor formation of a complex betweenchimeric immunoglobulin and human CD52 (e.g., a membrane fractioncomprising human CD52, on a cell bearing human CD52, such as a human Tcell, a human B cell, CHO cell or a recombinant host cell comprising anucleic acid encoding human CD52, a peptide (e.g., synthetic peptide)having an amino acid sequence of CD52).

Also provided herein are portions of the chimeric immunoglobulins whichinclude light chains, heavy chains and portions of light and heavychains. These immunoglobulin portions can be obtained or derived fromimmunoglobulins (e.g., by reduction and/or cleavage), or nucleic acidsencoding a portion of an immunoglobulin or chain thereof having thedesired property (e.g., binds human CD52, sequence similarity) can beproduced and expressed. They may be prepared by e.g., de novo synthesisof a portion. Chimeric immunoglobulins comprising the desired portions(e.g., antigen-binding region, CDR, FR, and/or C region) of human andnon-human origin can be produced using synthetic and/or recombinantnucleic acids to prepare constructs (e.g., cDNA) encoding the desiredchimeric chain. To prepare a portion of a chain, one or more stop codonscan be introduced at the desired position. A sequence encoding a signalpeptide can also be incorporated into the nucleic acid (e.g., onsynthesis, upon insertion into a vector). If the natural signal peptidesequence is unavailable (e.g., typically not present), a signal peptidesequence from another antibody can be used (see, e.g., Kettleborough, C.A., Protein Engineering 4:773-783 (1991)). Using these methods, methodsdescribed herein or other suitable methods, variants can be readilyproduced.

In one embodiment, a chimeric immunoglobulin of this invention comprisesthe light chain variable region of SEQ ID NO: 3 and the heavy chainvariable region of SEQ ID NO: 16; the light chain variable region of SEQID NO: 4 and the heavy chain variable region of SEQ ID NO: 17; the lightchain variable region of SEQ ID NO: 5 and the heavy chain variableregion of SEQ ID NO: 18; the light chain variable region of SEQ ID NO: 6and the heavy chain variable region of SEQ ID NO: 19; the light chainvariable region of SEQ ID NO: 7 and the heavy chain variable region ofSEQ ID NO: 20; the light chain variable region of SEQ ID NO: 8 and theheavy chain variable region of SEQ ID NO: 21; the light chain variableregion of SEQ ID NO: 9 and the heavy chain variable region of SEQ ID NO:22; the light chain variable region of SEQ ID NO: 10 and the heavy chainvariable region of SEQ ID NO: 23; the light chain variable region of SEQID NO: 11 and the heavy chain variable region of SEQ ID NO: 24; thelight chain variable region of SEQ ID NO: 12 and the heavy chainvariable region of SEQ ID NO: 25; or the light chain variable region ofSEQ ID NO: 13 and the heavy chain variable region of SEQ ID NO: 26.

The invention also relates to a chimeric antibody that has bindingspecificity for human CD52, comprising a light chain variable regionsequence selected from the group consisting of the light chain variableregion of SEQ ID NO: 3, the light chain variable region of SEQ ID NO: 4,the light chain variable region of SEQ ID NO: 5, the light chainvariable region of SEQ ID NO: 6, the light chain variable region of SEQID NO: 7, the light chain variable region of SEQ ID NO: 8, the lightchain variable region of SEQ ID NO: 9, the light chain variable regionof SEQ ID NO: 10, the light chain variable region of SEQ ID NO: 11, thelight chain variable region of SEQ ID NO: 12 and the light chainvariable region of SEQ ID NO: 13, and a heavy chain variable regionsequence selected from the group consisting of: the heavy chain variableregion of SEQ ID NO: 16, the heavy chain variable region of SEQ ID NO:17, the heavy chain variable region of SEQ ID NO: 18, the heavy chainvariable region of SEQ ID NO: 19, the heavy chain variable region of SEQID NO: 20, the heavy chain variable region of SEQ ID NO: 21, the heavychain variable region of SEQ ID NO: 22, the heavy chain variable regionof SEQ ID NO: 23, the heavy chain variable region of SEQ ID NO: 24, theheavy chain variable region of SEQ ID NO: 25 and the heavy chainvariable region of SEQ ID NO: 26.

The invention also relates to a chimeric light chain comprising thevariable region of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, or SEQ ID NO: 13.

The invention also relates to a chimeric heavy chain comprising thevariable region of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23; SEQID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26

If desired, for example, for diagnostic or assay purposes (e.g.,imaging), the chimeric immunoglobulin (e.g., antigen-binding fragmentthereof) can comprise a detectable label. Suitable detectable labels andmethods for labeling a chimeric immunoglobulin are well known in theart. Suitable detectable labels include, for example, a radioisotope(e.g., as Indium-111, Technnetium-99m or Iodine-131), positron emittinglabels (e.g., Fluorine-19), paramagnetic ions (e.g., Gadlinium (III),Manganese (II)), an epitope label (tag), an affinity label (e.g.,biotin, avidin), a spin label, an enzyme, a fluorescent group or achemiluminescent group. When labels are not employed, complex formation(e.g., between chimeric immunoglobulin and human CD52) can be determinedby surface plasmon resonance or other suitable methods. All suitablemethods and techniques described above for humanized antibodies of thisinvention can also be used herein.

Nucleic Acids and Recombinant Vectors

The present invention also relates to isolated and/or recombinant(including, e.g., essentially pure) nucleic acids comprising sequenceswhich encode a humanized immunoglobulin, humanized light chain,humanized heavy chain, mouse monoclonal immunoglobulin, mouseimmunoglobulin light chain, mouse immunoglobulin heavy chain, chimericimmunoglobulin, chimeric light chain or chimeric heavy chain of thepresent invention.

Nucleic acids referred to herein as “isolated” or “purified” are nucleicacids which have been separated away from the nucleic acids of thegenomic DNA or cellular RNA of their source of origin (e.g., as theyexist in cells or in a mixture of nucleic acids such as a library), andinclude nucleic acids obtained by methods described herein or othersuitable methods, including essentially pure nucleic acids, nucleicacids produced by chemical synthesis, by combinations of biological andchemical methods, and recombinant nucleic acids which are isolated (seee.g., Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476(1991); Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)).

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures which rely upon a methodof artificial recombination, such as the polymerase chain reaction (PCR)and/or cloning into a vector using restriction enzymes. “Recombinant”nucleic acids are also those that result from recombination events thatoccur through the natural mechanisms of cells, but are selected forafter the introduction to the cells of nucleic acids designed to allowand make probable a desired recombination event.

The present invention also relates more specifically to isolated and/orrecombinant nucleic acids comprising a nucleotide sequence which encodesa humanized immunoglobulin, mouse immunoglobulin or chimericimmunoglobulin that has binding specificity for human CD52 (e.g., ahumanized immunoglobulin of the present invention in which the nonhumanportion (e.g., the CDRs) is derived from a murine anti-CD52 monoclonalantibody; a mouse immunoglobulin of the present invention; or a chimericimmunoglobulin of the present invention in which the nonhuman portion(e.g., the V_(H) and V_(L)) is derived from a murine anti-CD52monoclonal antibody) or portion (e.g., antigen-binding portion) thereof(e.g., heavy or light chain thereof).

Nucleic acids of the present invention can be used to produce humanizedimmunoglobulins having binding specificity for human CD52, mouseimmunoglobulins having binding specificity for human CD52 and chimericimmunoglobulins having binding specificity for human CD52. For example,a nucleic acid (e.g., DNA (such as cDNA), or RNA) or one or more nucleicacids encoding a humanized immunoglobulin, mouse immunoglobulin orchimeric immunoglobulin of the present invention can be incorporatedinto a suitable construct (e.g., a recombinant vector) for furthermanipulation of sequences or for production of the encodedimmunoglobulins in suitable host cells.

Constructs or vectors (e.g., expression vectors) suitable for theexpression of a humanized immunoglobulin having binding specificity forhuman CD52, mouse immunoglobulin having binding specificity for humanCD52 or chimeric immunoglobulin having binding specificity for humanCD52 are also provided. A variety of vectors are available, includingvectors which are maintained in single copy or multiple copies in a hostcell, or which become integrated into the host cell's chromosome(s). Theconstructs or vectors can be introduced into a suitable host cell, andcells which express a humanized immunoglobulin, mouse immunoglobulin orchimeric immunoglobulin of the present invention, can be produced andmaintained in culture. A single vector or multiple vectors can be usedfor the expression of a humanized immunoglobulin, mouse immunoglobulinor chimeric immunoglobulin having binding specificity for human CD52.

Suitable expression vectors, for example mammalian cell expressionvectors, can also contain a number of components, including, but notlimited to one or more of the following: an origin of replication; aselectable marker gene; one or more expression control elements, such asa transcriptional control element (e.g., a promoter, an enhancer, aterminator), and/or one or more translation signals; a signal sequenceor leader sequence for membrane targeting or secretion. In a constructor vector, a signal peptide sequence can be provided by the construct orvector or other source. For example, the transcriptional and/ortranslational signals of an immunoglobulin can be used to directexpression.

A promoter can be provided for expression in a suitable host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding a humanized immunoglobulinor immunoglobulin chain, such that it directs expression of the encodedpolypeptide. A variety of suitable promoters for prokaryotic (e.g., lac,tac, T3, T7 promoters for E. coli) and eukaryotic (e.g., yeast alcoholdehydrogenase (ADH1), SV40, CMV) hosts are available. Those of skill inthe art will be able to select the appropriate promoter for expressingan anti-CD52 antibody or portion thereof of the invention.

In addition, the vectors (e.g., expression vectors) typically comprise aselectable marker for selection of host cells carrying the vector, and,in the case of a replicable vector, an origin of replication. Genesencoding products which confer antibiotic or drug resistance are commonselectable markers and may be used in prokaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene (tetracycline resistance) andeukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated.

The invention thus relates to isolated nucleic acid molecules thatencode the humanized immunoglobulin, humanized light chain, humanizedheavy chain, mouse immunoglobulin, mouse immunoglobuin light chain,mouse immunoglobuin heavy chain, chimeric immunoglobulin, chimeric lightchain, or chimeric heavy chain of this invention. The invention alsorelates to isolated nucleic acid molecules that encode anantigen-binding portion of the immunoglobulins and their chains.Polypeptide sequences encoded by the nucleic acids of this invention aredescribed above and in the following Examples.

In some embodiments, a nucleic acid and vector of this invention encodesa heavy chain (or an antigen-binding portion thereof) or a light chain(or an antigen-binding portion thereof) of this invention. A host cellcontaining both the heavy chain-encoding nucleic acid and the lightchain-encoding nucleic acid can be used to make an antibody comprisingthe heavy and light chain (or an antigen-binding portion of theantibody). The heavy chain-encoding nucleic acid and the lightchain-encoding nucleic acid can be placed on separate expressionvectors. They can also be placed on a single expression vector under thesame or different expression control. See, e.g., Cabilly U.S. Pat. No.6,331,415; Fang U.S. Pat. No. 7,662,623.

Method of Producing Immunoglobulins Having Specificity for Human CD52

Another aspect of the invention relates to a method of making ananti-human CD52 antibody of this invention. The antibody of thisinvention can be produced, for example, by the expression of one or morerecombinant nucleic acids encoding the antibody in a suitable host cell.The host cell can be produced using any suitable method. For example,the expression constructs (e.g., the one or more vectors, e.g., amammalian cell expression vector) described herein can be introducedinto a suitable host cell, and the resulting cell can be maintained(e.g., in culture, in an animal, in a plant) under conditions suitablefor expression of the construct(s) or vector(s). Suitable host cells canbe prokaryotic, including bacterial cells such as E. coli (e.g., strainDH5α™ (Invitrogen, Carlsbad, Calif.)), B. subtilis and/or other suitablebacteria; eukaryotic cells, such as fungal or yeast cells (e.g., Pichiapastoris, Aspergillus sp., Saccharomyces cerevisiae, Schizosaccharomycespombe, Neurospora crassa), or other lower eukaryotic cells, and cells ofhigher eukaryotes such as those from insects (e.g., Drosophila SchniederS2 cells, Sf9 insect cells (WO 94/26087 (O'Connor), TN5B1-4 (HIGH 5)insect cells (Invitrogen), mammals (e.g., COS cells, such as COS-1 (ATCCAccession No. CRL-1650) and COS-7 (ATCC Accession No. CRL-1651), CHO(e.g., ATCC Accession No. CRL-9096), CHO DG44 (Urlaub, G. and Chasin, LA., Proc. Natl. Acac. Sci. USA, 77(7):4216-4220 (1980)), 293 (ATCCAccession No. CRL-1573), HeLa (ATCC Accession No. CCL-2), CV1 (ATCCAccession No. CCL-70), WOP (Dailey, L., et al., J. Virol., 54:739-749(1985)), 3T3, 293T (Pear, W. S., et al., Proc. Natl. Acad. Sci. U.S.A.,90:8392-8396 (1993)), NS0 cells, SP2/0 cells, HuT 78 cells and thelike)), or plants (e.g., tobacco, lemna (duckweed), and algae). (See,for example, Ausubel, F. M. et al., eds. Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons Inc.(1993)). In some embodiments, the host cell is not part of amulticellular organism (e.g., plant or animal), e.g., it is an isolatedhost cell or is part of a cell culture.

The present invention also relates to cells comprising a nucleic acid,e.g., a vector, of the invention (e.g., an expression vector). Forexample, a nucleic acid (i.e., one or more nucleic acids) encoding theheavy and light chains of a humanized immunoglobulin, the heavy andlight chains of mouse immunoglobulin, or the heavy and light chains of achimeric immunoglobulin, said immunoglobulin having binding specificityfor human CD52, or a construct (i.e., one or more constructs, e.g., oneor more vectors) comprising such nucleic acid(s), can be introduced intoa suitable host cell by a method appropriate to the host cell selected(e.g., transformation, transfection, electroporation, infection), withthe nucleic acid(s) being, or becoming, operably linked to one or moreexpression control elements (e.g., in a vector, in a construct createdby processes in the cell, integrated into the host cell genome). Hostcells can be maintained under conditions suitable for expression (e.g.,in the presence of inducer, suitable media supplemented with appropriatesalts, growth factors, antibiotic, nutritional supplements, etc.),whereby the encoded polypeptide(s) are produced. If desired, the encodedprotein (e.g., humanized immunoglobulin, mouse immunoglobulin, chimericimmunoglobulin) can be isolated, for example, from the host cells,culture medium, or milk. This process encompasses expression in a hostcell (e.g., a mammary gland cell) of a transgenic animal or plant (e.g.,tobacco) (see e.g., WO 92/03918).

Fusion proteins can be produced in which an immunoglobulin portion(e.g., a humanized immunoglobulin; immunoglobulin chain) is linked to anon-immunoglobulin moiety (i.e., a moiety which does not occur inimmunoglobulins as found in nature) in an N-terminal location,C-terminal location or internal to the fusion protein. For example, someembodiments can be produced by the insertion of a nucleic acid encodingan immunoglobulin sequence(s) into a suitable expression vector, such asa pET vector (e.g., pET-15b, Novagen), a phage vector (e.g., pCANTAB 5E, Pharmacia), or other vector (e.g., pRIT2T Protein A fusion vector,Pharmacia). The resulting construct can be introduced into a suitablehost cell for expression. Upon expression, some fusion proteins can beisolated or purified from a cell lysate by means of a suitable affinitymatrix (see, e.g., Current Protocols in Molecular Biology (Ausubel, F.M. et al., Eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).

The invention relates to a host cell that comprises recombinant nucleicacid(s) encoding an immunoglobulin provided herein (e.g., a humanizedimmunoglobulin, a humanized light chain or a humanized heavy chain, amouse immunoglobulin, a mouse light chain or a mouse heavy chain, achimeric immunoglobulin, a chimeric heavy chain, or a chimeric lightchain of the invention). The invention also relates to a host cell thatcomprises recombinant nucleic acid(s) encoding an antigen-bindingportion of the immunoglobulin or their chains. In some embodiments, thehost cell comprises a recombinant vector (e.g., expression vector,mammalian cell expression vector) of the invention as referred toherein.

The invention also relates to a method of preparing an immunoglobulin oran immunoglobulin polypeptide chain of this invention. In oneembodiment, the method comprises maintaining a host cell of theinvention as described herein (e.g., a host cell that contains one ormore isolated nucleic acids that encode the immunoglobulin orpolypeptide chain (e.g., a light chain and a heavy chain, a light chainonly, or a heavy chain only, of the invention) under conditionsappropriate for expression of the immunoglobulin or polypeptide chain.For example a host cell can be cultured on a substrate or in suspension.In some embodiments, the method further comprises the step of purifyingor isolating the immunoglobulin or polypeptide chain.

The invention further relates to a method of preparing immunoglobulinsthrough phage display. For example, a naïve antibody phage displaylibrary on CD52 antigen can be panned. Alternatively, a method ofpreparing immunoglobulins through guided selection can be used (U.S.Patent Application Publication US 2006-0251658 A1.) A custom librarybuilt around, for example, a fixed heavy chain (and/or light chain) CDR3region of a known anti-CD52 antibody can be created. The CDR1 and CDR2regions of the heavy and light chains can be derived from a naïverepertiore (Osburn et al., Methods, 36:61-68 (2005)). In one embodiment,anti-CD52 ScFvs can be generated from ScFv naïve antibody librarieswhich are used to obtain mouse-human chimeric antibodies with thedesired binding properties. These libraries may be screened forantibodies with the desired binding properties. ScFv phage libraries maybe used. For example, ScFvs which recognize human CD52 can be isolatedfrom scFv guided selection libraries following a series of repeatedselection cycles on recombinant human CD52 essentially as described inVaughan et al. (1996). In brief, following incubation with the library,the immobilized antigen, which is pre-coupled to paramagnetic beads, andbound phage can be recovered by magnetic separation while unbound phageis washed away. Bound phage can then be rescued as described by Vaughanet al. (1996) and the selection process repeated.

In a particular embodiment, a library is constructed consisting of theentire variable domain of the heavy chain of a mouse anti-CD52 antibodyfused in a single chain format to a repertoire of naive human lightchain variable regions. After selection the pool of human light chainvariable regions that complement the mouse heavy chain variable regionare identified. A library is then constructed consisting of therepertoire of human light chain variable regions selected above fused ina single chain format to a chimeric heavy chain variable regionconsisting of naive human CDR1 and CDR2 regions and a fixed CDR3 regionfrom the mouse anti-CD52 antibody heavy chain variable domain. Afterselection for CD52 binders, the best binding clones are selected. Fiveof the 6 CDR regions can be human in origin while the CDR-3 of the heavychain variable region can be identical to the original CDR3 of the mouseheavy chain variable domain.

Selections can be performed using CD52 coupled to DYNABEADS M-270 amine(Dynal) according to the manufacturer's recommendations. Alternatively,selections using biotinylated CD52 can be prepared using the primaryamine specific reagent succinimidyl-6-(biotinamido)hexanoate followingthe manufacturer's instructions (EZ link NHS LC Biotin, Pierce).

Outputs from selections can be tested as periplasmic preparations inhigh throughput screens based on competition assays which measure theability of the scFvs present in the periplasmic preparation to competefor binding to CD52.

Samples that are able to compete in the high throughput screens may besubjected to DNA sequencing as described in Vaughan et al. (1996) andOsburn et al. (1996). Clones would then be expressed and purified asscFvs or IgGs and assessed for their ability to bind CD52, neutralizeCD52 or a combination thereof, e.g., using assays such asantibody-dependent cell mediated cytotoxicity (ADCC) assay andcomplement dependent cytotoxicity (CDC) assay. Purified scFvpreparations can then be prepared as described in Example 3 of WO01/66754. Protein concentrations of purified scFv preparations weredetermined using the BCA method (Pierce). Similar approaches can be usedto screen for an optimal partner (the opposite chain) of a fixedimmunoglobulin heavy or light chain (or V_(H) or V_(L)).

In a particular embodiment, the invention is directed to a method ofproducing a hybridoma that secretes a monoclonal antibody that hasbinding specificity for human CD52 comprising administering lymphocytesof a CD52 transgenic mouse to a non-transgenic mouse having the samestrain (e.g., CD1) as the human CD52 transgenic mouse, thereby producingan immunized, non-transgenic mouse. Splenocytes of the immunized,non-transgenic mouse are contacted with immortalized cells, therebyproducing fused cells, and the fused cells are maintained underconditions in which hybridomas that secrete a monoclonal antibody havingbinding specificity for human CD52 are produced, thereby producing ahybridoma that secretes a monoclonal antibody that has bindingspecificity for human CD52.

Immunoglobulins Containing a Toxin Moiety or Toxin

The invention also relates to immunoglobulins that comprise a toxinmoiety or toxin. Suitable toxin moieties comprise a toxin (e.g., surfaceactive toxin, cytotoxin). The toxin moiety or toxin can be linked orconjugated to the immunoglobulin using any suitable method. For example,the toxin moiety or toxin can be covalently bonded to the immunoglobulindirectly or through a suitable linker. Suitable linkers can includenoncleavable or cleavable linkers, for example, pH cleavable linkers orlinkers that comprise a cleavage site for a cellular enzyme (e.g.,cellular esterases, cellular proteases such as cathepsin B). Suchcleavable linkers can be used to prepare an immunoglobulin that canrelease a toxin moiety or toxin after the immunoglobulin isinternalized.

A variety of methods for linking or conjugating a toxin moiety or toxinto an immunoglobulin can be used. The particular method selected willdepend on the toxin moiety or toxin and immunoglobulin to be linked orconjugated. If desired, linkers that contain terminal functional groupscan be used to link the immunoglobulin and toxin moiety or toxin.Generally, conjugation is accomplished by reacting toxin moiety or toxinthat contains a reactive functional group (or is modified to contain areactive functional group) with a linker or directly with animmunoglobulin. Covalent bonds are formed by reacting a toxin moiety ortoxin that contains (or is modified to contain) a chemical moiety orfunctional group that can, under appropriate conditions, react with asecond chemical group thereby forming a covalent bond. If desired, asuitable reactive chemical group can be added to an immunoglobulin or toa linker using any suitable method. (See, e.g., Hermanson, G. T.,Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).) Manysuitable reactive chemical group combinations are known in the art, forexample an amine group can react with an electrophilic group such astosylate, mesylate, halo (chloro, bromo, fluoro, iodo),N-hydroxysuccinimidyl ester (NHS), and the like. Thiols can react withmaleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)).

Suitable toxin moieties and toxins include, for example, a maytansinoid(e.g., maytansinol, e.g., DM1, DM4), a taxane, a calicheamicin, aduocarmycin, or derivatives thereof. The maytansinoid can be, forexample, maytansinol or a maytansinol analogue. Examples of maytansinolanalogs include those having a modified aromatic ring (e.g.,C-19-decloro, C-20-demethoxy, C-20-acyloxy) and those havingmodifications at other positions (e.g., C-9-CH, C-14-alkoxymethyl,C-14-hydroxymethyl or aceloxymethyl, C-15-hydroxy/acyloxy, C-15-methoxy,C-18-N-demethyl, 4,5-deoxy). Maytansinol and maytansinol analogs aredescribed, for example, in U.S. Pat. Nos. 5,208,020 and 6,333,410, thecontents of which are incorporated herein by reference. Maytansinol canbe coupled to antibodies and antibody fragments using, e.g., anN-succinimidyl 3-(2-pyridyldithio)proprionate (also known asN-succinimidyl 4-(2-pyridyldithio)pentanoate (or SPP)),4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)-toluene (SMPT),N-succinimidyl-3-(2-pyridyldithio)butyrate (SDPB), 2 iminothiolane, orS-acetylsuccinic anhydride. The taxane can be, for example, a taxol,taxotere, or novel taxane (see, e.g., WO 01/38318). The calicheamicincan be, for example, a bromo-complex calicheamicin (e.g., an alpha, betaor gamma bromo-complex), an iodo-complex calicheamicin (e.g., an alpha,beta or gamma iodo-complex), or analogs and mimics thereof.Bromo-complex calicheamicins include I1-BR, I2-BR, I3-BR, I4-BR, J1-BR,J2-BR and K1-BR. Iodo-complex calicheamicins include I1-I, I2-I, I3-I,J1-I, J2-I, L1-I and K1-BR. Calicheamicin and mutants, analogs andmimics thereof are described, for example, in U.S. Pat. Nos. 4,970,198;5,264,586; 5,550,246; 5,712,374, and 5,714,586, the contents of each ofwhich are incorporated herein by reference. Duocarmycin analogs (e.g.,KW-2189, DC88, DC89 CBI-TMI, and derivatives thereof) are described, forexample, in U.S. Pat. No. 5,070,092, U.S. Pat. No. 5,187,186, U.S. Pat.No. 5,641,780, U.S. Pat. No. 5,641,780, U.S. Pat. No. 4,923,990, andU.S. Pat. No. 5,101,038, the contents of each of which are incorporatedherein by reference.

Examples of other toxins include, but are not limited to antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499,5,846,545), melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, mitomycin, puromycin anthramycin (AMC)),duocarmycin and analogs or derivatives thereof, and anti-mitotic agents(e.g., vincristine, vinblastine, taxol, auristatins (e.g., auristatin E)and maytansinoids, and analogs or homologs thereof).

The toxin can also be a surface active toxin, such as a toxin that is afree radical generator (e.g., selenium containing toxin moieties), orradionuclide containing moiety. Suitable radionuclide containingmoieties, include for example, moieties that contain radioactive iodine(131I or 125I), yttrium (90Y), lutetium (177Lu), actinium (225Ac),praseodymium, astatine (211At), rhenium (186Re), bismuth (212Bi or213Bi), indium (111In), technetium (99mTc), phosphorus (32P), rhodium(188Rh), sulfur (35S), carbon (14C), tritium (3H), chromium (51Cr),chlorine (36Cl), cobalt (57Co or 58Co), iron (59Fe), selenium (75Se), orgallium (67Ga).

The toxin can be a protein, polypeptide or peptide, from bacterialsources, e.g., diphtheria toxin, pseudomonas exotoxin (PE) and plantproteins, e.g., the A chain of ricin (RTA), the ribosome inactivatingproteins (RIPs) gelonin, pokeweed antiviral protein, saporin, anddodecandron are contemplated for use as toxins.

Antisense compounds of nucleic acids designed to bind, disable, promotedegradation or prevent the production of the mRNA responsible forgenerating a particular target protein can also be used as a toxin.Antisense compounds include antisense RNA or DNA, single or doublestranded, oligonucleotides, or their analogs, which can hybridizespecifically to individual mRNA species and prevent transcription and/orRNA processing of the mRNA species and/or translation of the encodedpolypeptide and thereby effect a reduction in the amount of therespective encoded polypeptide. Ching, et al., Proc. Natl. Acad. Sci.U.S.A. 86: 10006-10010 (1989); Broder, et al., Ann. Int. Med. 113:604-618 (1990); Loreau, et al., FEBS Letters 274: 53-56 (1990). Usefulantisense therapeutics include for example: Veglin™ (VasGene) andOGX-011 (Oncogenix).

Toxins can also be photoactive agents. Suitable photoactive agentsinclude porphyrin-based materials such as porfimer sodium, the greenporphyrins, chlorin E6, hematoporphyrin derivative itself,phthalocyanines, etiopurpurins, texaphrin, and the like.

The toxin can be an antibody or antibody fragment that binds anintracellular target. Such antibodies or antibody fragments can bedirected to defined subcellular compartments or targets. For example,the antibodies or antibody fragments can bind an intracellular targetselected from erbB2, EGFR, BCR-ABL, p21Ras, Caspase3, Caspase7, Bcl-2,p53, Cyclin E, ATF-1/CREB, HPV16 E7, HP1, Type IV collagenases,cathepsin L as well as others described in Kontermann, R. E., Methods,34:163-170 (2004), incorporated herein by reference in its entirety.

Therapeutic Methods and Compositions

The antibodies of this invention are useful in immuno-suppression andimmuno-ablation. The antibodies target CD52-expressing cells (e.g., Tand B cells) and reduce (or “deplete” as used herein) their populationin a subject in need thereof. Lymphocyte depletion may be useful intreating a variety of diseases and conditions such as inflammation,autoimmune diseases and cancer (e.g., lymphocyte (either B or T cell)malignancy) See, e.g., Reiff, A., Hematology, 10(2):79-93 (2005).Examples of diseases and conditions that can be treated with theantibodies or antigen-binding portions of this invention include,without limitation, multiple sclerosis, lupus, rheumatoid arthritis,graft versus host disease (GVHD), inflammatory bowl disease, vasculitis,Behcet's disease, Wegener's granulomatosis, Sjogren's syndrome, uveitis,psoriasis, scleroderma, polymyositis, type I (autoimmune-based)diabetes, autoimmune cytopenias (e.g., autoimmune neutropenia,transfusion-dependent refractory PRCA, leukemia and lymphoma such asnon-Hodgkin's lymphoma with bulky disease and B-cell chronic lymphocyticleukemia.

Accordingly, aspects of this invention are methods for lymphocytedepletion, and for treating inflammation, an autoimmune disease orcancer by administering an effective amount of an antibody of theinvention to a subject in need thereof (e.g., a human patient having anautoimmune disease, a blood cancer, or a patient who is to receive atransplantation). The antibody also can be administered prophylacticallyto prevent onset of inflammation or relapse of an autoimmune disease orcancer. For example, the antibody of this invention can be administeredas part of a conditioning regimen to prepare a patient for atransplantation (e.g., a stem cell transplant, an infusion of autologousof allogeneic T cells, or a solid organ transplant).

Some anti-CD52 antibodies of this invention preferentially targetcertain populations of CD52+ cells. One possible explanation is thatepitopes to which these antibodies bind include one or more carbohydrategroups on the CD52 protein, and such carbohydrate groups may be moreprevalent on CD52 expressed on one cell type versus another. Forexample, we have found that antibody 7F11, 5F7, 3G7, and 11C11 deplete Tcells to a greater extent than B cells. Thus, the humanized and chimericversions of these antibodies may be used to treat T cell malignancy withmilder immunosuppressing side effects.

Because antibodies of this invention target CD52-expressing cells, theantibodies also can be used to deplete CD52+ cell types other than Tcells and B cells. For example, studies have shown that vascularleukocytes (VLC) and Tie2+ monocytes—myeloid cells expressing highlevels of CD52—promote tumor angiogenesis and contribute to tumorresistance to anti-VEGF therapy. Pulaski et al., J. Translational Med.7:49 (2009). Anti-CD52 antibodies of this invention thus can be used toinhibit tumor angiogenesis by targeting VLC and Tie2+ monocytes. Forthis purpose, the anti-CD52 antibodies can be administered systemically,or locally at a site of neovascularization, such as a tumor site.Anti-CD52 antibody therapy can be used in conjunction withstandard-of-care cancer treatment such as chemotherapy, surgery, orradiation, or with another targeted therapy such as anti-VEGF antibodytherapy. Anti-CD52 antibody therapy can be used to treat, for example,breast cancer, lung cancer, glioma, colorectal cancer, and any otherindications of anti-VEGF antibodies. Anti-CD52 antibody therapy also canbe used in other neovascularization conditions including non-oncologicalneovascular conditions.

Antibodies of this invention can be administered to an individual (e.g.,a human) alone or in conjunction with another agent (e.g., animmunosuppressant) in a combination therapy. The antibody can beadministered before, along with or subsequent to administration of theadditional agent. In some embodiments, the additional agent is, forexample, an anti-inflammatory compound such as sulfasalazine, anothernon-steroidal anti-inflammatory compound, or a steroidalanti-inflammatory compound. In some embodiments, the additional agent isanother lympho-depleting antibody such as another anti-CD52 antibody, ananti-CD20 antibody, an anti-BAFF antibody, an anti-BAFF-R antibody, andthe like. In some embodiments, the additional agent is, e.g., a cytokine(e.g., IL-7), anti-cytokine receptor antibody, or a soluble receptor,that skews, manipulates, and/or augments the reconstitution process thatoccurs following lymphodepletion mediated by an anti-CD52 antibody (see,e.g., Sportes et al., “ ”Cytokine Therapies: Ann. N.Y. Acad. Sci.1182:28-38 (2009)). In another embodiment, a synthetic peptide mimeticcan be administered in conjunction with an immunoglobulin of the presentinvention.

Studies have shown that lymphocyte depletion by alemtuzumab is mediatedby neutrophils and NK cells (Hu et al., Immunology 128:260-270 (2009).Thus, in an embodiment of combination therapy, an agent that stimulatesneutrophils and NK cells can be administered to a patient, before,during or after anti-CD52 antibody therapy, to augment the antibodytherapy. Stimulating neutrophils and/or NK cells include, withoutlimitation, (1) increasing their rates of division, (2) increasing theircell surface expression of the Fc receptors corresponding to the isotypeof the anti-CD52 antibody (e.g., FcγRIIIa and FcγRIIIb, FcγRII, FcγRI,and FcαRI), (3) mobilizing and increasing the number of circulatingcells, (4) recruiting the cells to target sites (e.g., sites of tumors,inflammation, or tissue damage), (5) and increasing their cytotoxicactivity. Examples of agents that stimulate neutrophils and/or NK cellsinclude, for example, granulocyte monocyte colony stimulating factor(GM-CSF) (e.g., LEUKINE® or sargramostim and molgramostim); granulocytecolony stimulating factor (G-CSF) (e.g., NEUPOGEN® or filgrastim,pegylated filgrastim, and lenograstim); interferon gamma (e.g.,ACTIMMUNE®); CXC chemokine receptor 4 (CXCR4) antagonists (e.g.,MOZOBIL™ or plerixafor); and CXC chemokine receptor 2 (CXCR2) agonists.The neutrophil count of the patient may be monitored periodically toensure optimal treatment efficacy. The neutrophil count of the patientalso can be measured prior to the start of the anti-CD52 antibodytreatment. The stimulator's amount can be adjusted based on thepatient's neutrophil count. A higher dose of the stimulator may be usedif the patient has a lower than normal neutrophil count. During periodsof neutropenia, which may be caused by treatment with the anti-CD52antibody, a higher dose of the neutrophil stimulator may also beadministered to maximize the effect of the anti-CD52 antibody.

Because neutrophil and/or NK stimulation improves the efficacy ofanti-CD52 antibody therapy, this embodiment of combination therapyallows one to use less antibody in a patient while maintaining similartreatment efficacy. Using less anti-CD52 antibody while maintainingtreatment efficacy may help reduce side effects of the anti-CD52antibody, which include immune response in the patient against theadministered antibody as well as development of secondary autoimmunity(autoimmunity that arises during or after anti-CD52 antibody treatment).This embodiment of combination of therapy is also useful in an oncologysetting, e.g., when the patient has neutropenia.

In another embodiment of combination therapy, one can use a stimulatorof regulatory T cells to augment anti-CD52 antibody therapy. Our datashow that anti-CD52 antibodies deplete CD4⁺CD25⁺FoxP3⁺ regulatory Tcells to a much lesser extent as compared to other CD4⁺ T cells.Regulatory T cells (also known as “Treg” or suppressor T cells) arecells that are capable of inhibiting the proliferation and/or functionof other lymphoid cells via contact-dependent or contact-independent(e.g., cytokine production) mechanisms. Several types of regulatory Tcells have been described, including γδ T cells, natural killer T (NKT)cells, CD8⁺T cells, CD4⁺T cells, and double negative CD4⁻CD8⁻T cells.See, e.g., Bach et al., Immunol. 3:189-98 (2003). CD4⁺CD25⁺FoxP3⁺regulatory T cells have been referred as “naturally occurring”regulatory T cells; they express CD4, CD25 and forkhead familytranscription factor FoxP3 (forkhead box p3). Thus, in this embodimentof combination therapy, one can administer an agent that stimulatesCD4⁺CD25⁺FoxP3⁺ regulatory T cells before, during or after the anti-CD52antibody therapy, to skew the composition of the immune system followinglympho-depletion. The agent may, for example, activate those T cells,stabilize and/or expand the population of the cells, mobilize andincrease circulation of the cells, and/or recruit the cells to targetsites. Examples of such agents are rapamycin, active or latent TGF-β(e.g., TGF-β1, TGF-β2, TGF-β3, TGF-β4, and TGF-β5), IL-10, IL-4, IFN-α,vitamin D3, dexamethasone, and mycophenolate mofetil (see, e.g., Barratet al., J. Exp. Med. 195:603-616 (2002); Gregori et al., J Immunol. 167:1945-1953 (2001); Battaglia et al., Blood 105: 4743-4748 (2005);Battaglia et al., J. Immunol. 177:8338-8347 (2006)).

In this invention, an effective amount of anti-CD52 antibody fortreating a disease is an amount that helps the treated subject to reachone or more desired clinical end points. For example, for lupus (whosemanifestations include systemic lupus erythematosus, lupus nephritis,cutaneous lupus erythematosus, CNS lupus, cardiovascular manifestations,pulmonary manifestations, hepatic manifestations, haematologicalmanifestations, gastrointestinal manifestations, musculoskeletalmanifestations, neonatal lupus erythematosus, childhood systemic lupuserythematosus, drug-induced lupus erythematosus, anti-phospholipidsyndrome, and complement deficiency syndromes resulting in lupusmanifestations; see, e.g., Robert G. Lahita, Editor, Systemic LupusErythematosus, 4th Ed., Elsevier Academic Press, 2004), clinicalendpoints can be measured by monitoring of an affected organ system(e.g., hematuria and/or proteinuria for lupus nephritis) and/or using adisease activity index that provides a composite score of diseaseseverity across several organ systems (e.g., BILAG, SLAM, SLEDAI,ECLAM). See, e.g., Mandl et al., “Monitoring patients with systemiclupus erythematosus” in Systemic Lupus Erythematosus, 4^(th) edition,pp. 619-631, R. G. Lahita, Editor, Elsevier Academic Press, (2004).

In another example of autoimmune disease, multiple sclerosis (includingrelapsing-remitting, secondary progressive, primary progressive, andprogressive relapsing multiple sclerosis ((Lublin et al., Neurology 46(4), 907-11 (1996)), diagnosed is made by, for example, the history ofsymptoms and neurological examination with the help of tests such asmagnetic resonance imaging (MRI), spinal taps, evoked potential tests,and laboratory analysis of blood samples. In MS, the goal of treatmentis to reduce the frequency and severity of relapses, prevent disabilityarising from disease progression, and promote tissue repair (Compstonand Coles, 2008). Thus, an amount of anti-CD52 antibody that helpsachieve a clinical endpoint consistent with that goal is an effectiveamount of antibody for the treatment.

To minimize immunogenicity, it is preferred that a humanized antibody beused to treat a human patient in therapeutic methods and compositions ofthis invention. In cases where repeated administration is not necessary,it may also be appropriate to administer a mouse:human chimeric antibodyof this invention to a human patient.

The antibodies of the invention can be used to treat an individual whohas previously been treated with Campath-1H® who has developedneutralizing antibodies to Campath-1H® (e.g., a Campath-1H®-refractoryindividual). For example, one could treat an individual having anautoimmune disease (e.g., multiple sclerosis, lupus, vasculitis) and/ora cancer (e.g., a leukemia (e.g., chronic lymphocytic leukemia), alymphoma (e.g., non-Hodgkin's lymphoma)) who has previously been treatedwith Campath-1H® (e.g., with one or more courses of Campath-1H®treatment) and who has developed neutralizing antibodies to Campath-1H®that reduce the efficacy of further Campath-1H® treatment. We have shownthat the humanized antibodies of this invention (e.g., humanized 2C3,12G6, and 9D9) can bind to human CD52 despite the presence ofneutralizing antibodies to alemtuzumab. In another embodiment, one couldtreat an individual who had become refractory to treatment with aparticular humanized antibody described herein with one of the otherhumanized antibodies described herein.

The antibody of this invention can be administered in a single unit doseor multiple doses at any time point deemed appropriate by a health careprovider. The dosage can be determined by methods known in the art andcan be dependent, for example, upon the individual's age, sensitivity,tolerance and overall well-being. A variety of routes of administrationcan be used, including, but not necessarily limited to, parenteral(e.g., intravenous, intraarterial, intramuscular, intrathecal,intraperitoneal, subcutaneous injection), oral (e.g., dietary), locally,topical, inhalation (e.g., intrabronchial, intranasal or oralinhalation, intranasal drops), or rectal, depending on the disease orcondition to be treated. Parenteral administration may be one preferredmode of administration.

In one embodiment, the antibodies of the invention are administered to apatient using the same dosing regimen as Campath-1H® (e.g., the dosingregimen of Campath-1H® for chronic lymphocytic leukemia). In anotherembodiment, an antibody of the invention is administered to a patienthaving an autoimmune disease (e.g., multiple sclerosis (MS)) in aregimen comprising administration of a first cycle of the antibodyfollowed by at least one further cycle of the antibody, in which eachtreatment cycle comprises 1-5 doses that are applied on consecutivedays, and wherein each treatment cycle is separated from the next cycleby at least 1-24 months (e.g., 12 months). For example, in oneembodiment, a patient having multiple sclerosis is treated with a firstcycle of the antibody comprising 5 daily doses of the antibody followedby at least one further cycle of antibody treatment, in which thetreatment occurs 12 months after the first cycle and comprises 3 dosesof the antibody applied on consecutive days. In another embodiment, apatients having MS is only re-treated once evidence of renewed MSactivity has been observed (see, e.g., WO 2008/031626; the teachings ofwhich are incorporated herein by reference in their entirety). In someembodiments, it may be necessary to administer more frequent courses oftreatment (e.g., every four months, every six months) if patients withmore advanced forms of MS or more progressive forms of other autoimmunediseases (such as vasculitis; see, e.g., Walsh et al., Ann Rheum Dis67:1322-1327 (2008)) experience a relapse early on after their lastcourse of treatment. Evidence of renewed MS activity may be determinedbased on the professional judgment of the treating clinician, using anymeans that may be available to such clinician. A variety of techniquesare currently available to clinicians to diagnose renewed MS activityincluding, without limitation, by clinical means (relapse or progressionof neurological disability) or by magnetic resonance imaging (MRI) ofthe brain or spinal cord. As is well understood by medicalpractitioners, disease activity detected via MRI may be indicated by theoccurrence of new cerebral or spinal lesions on T1 (enhanced ornon-enhanced)- or T2-weighted images or by the increase of the volume ofsuch lesions. As diagnostic methods for MS are continually evolving, itis anticipated there may be additional methods in the future that willdetect renewed MS activity (e.g., magnetization transfer ratio orMR-spectroscopy). The particular diagnostic method used to detectrenewed MS activity is not a limitation of the claimed invention. Incertain embodiments, repeated MRIs are performed in fixed intervalsafter a treatment cycle in order to determine whether re-treatment ofany given patient is necessary and the optimal time point forre-treatment of such patient. In general, it is desirable forre-treatment to occur before the disease re-manifests clinically.

Formulation will vary according to the route of administration selected(e.g., solution, emulsion). An appropriate composition comprising theantibody to be administered can be prepared in a physiologicallyacceptable vehicle or carrier. The composition can comprise multipledoses or be a single unit dose composition. For solutions or emulsions,suitable carriers include, for example, aqueous or alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles can include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles can include various additives,preservatives, or fluid, nutrient or electrolyte replenishers (See,generally, Remington's Pharmaceutical Sciences, 17th Edition, MackPublishing Co., PA, 1985). For inhalation, the compound can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

Diagnostic Methods and Compositions

The immunoglobulins of the present invention also are useful in avariety of processes with applications in research and diagnosis. Forinstance, they can be used to detect, isolate, and/or purify human CD52or variants thereof (e.g., by affinity purification or other suitablemethods such as flow cytometry, e.g., for cells, such as lymphocytes, insuspension), and to study human CD52 structure (e.g., conformation) andfunction. For in vitro applications, wherein immunogenicity of theantibody is not a concern, the mouse and chimeric antibodies of thisinvention will be useful in addition to humanized antibodies.

The immunoglobulins of the present invention can be used in diagnosticapplications (e.g., in vitro, ex vivo). For example, the humanizedimmunoglobulins of the present invention can be used to detect and/ormeasure the level of human CD52 in a sample (e.g., on cells expressinghuman CD52 in tissues or body fluids, such as an inflammatory exudate,blood, serum, bowel fluid, tissues bearing human CD52). A sample (e.g.,tissue and/or body fluid) can be obtained from an individual and animmunoglobulin described herein can be used in a suitable immunologicalmethod to detect and/or measure human CD52 expression, including methodssuch as flow cytometry (e.g., for cells in suspension such aslymphocytes), enzyme-linked immunosorbent assays (ELISA), includingchemiluminescence assays, radioimmunoassay, and immunohistology.

In one embodiment, a method of detecting human CD52 in a sample isprovided, comprising contacting a sample with an immunoglobulin of thepresent invention under conditions suitable for specific binding of theimmunoglobulin to human CD52 and detecting antibody-CD52 complexes whichare formed. In an application of the method, the immunoglobulinsdescribed herein can be used to analyze normal versus inflamed tissues(e.g., from a human) for human CD52 reactivity and/or expression (e.g.,immunohistologically) to detect associations between e.g., inflammatorybowel disease (IBD), autoimmune diseases (such as multiple sclerosis andlupus), cancer (such as non-Hodgkin's lymphoma and chronic lymphocyticleukemia), or other conditions and increased expression of human CD52(e.g., in affected tissues). Thus, the immunoglobulins of the presentinvention permit immunological methods of assessment of the presence ofhuman CD52 in normal and inflamed tissues, through which the presence ofdisease, disease progress and/or the efficacy of anti-human CD52 therapyin the treatment of disease, e.g., inflammatory disease can be assessed.

In addition, the immunoglobulins can be used to examine tissues aftertreatment with a depleting anti-CD52 therapeutic antibody to gauge howeffective the depletion has been as well as to determine whether therehas been any downregulation in the expression of CD52 (Rawstrom et al.,Br. J. Heam., 107:148-153 (1999)).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Although a number of documents are cited herein, this citationdoes not constitute an admission that any of these documents forms partof the common general knowledge in the art. Throughout thisspecification and claims, the word “comprise,” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or group of integers but not the exclusion of any otherinteger or group of integers. The materials, methods, and examples areillustrative only and not intended to be limiting.

EXEMPLIFICATION Example 1 Generation of Mouse Anti-Human CD52 Antibodies

The mouse anti-human CD52 antibodies in the following working exampleswere generated by immunizing CD1 strain mice with splenocytes from humanCD52 transgenic mice with a CD1 background (FIG. 1A), where display ofhuman CD52 on the surface of mouse B and T cells of the transgenic micewas verified by flow cytometry. Because the transgenic mice had the samebackground (CD1) as the immunized mice, splenocytes from the transgenicmice presented human CD52 at the cell surface as a unique, non-selfantigen in a native format, and the immunized nontransgenic mice mountedan antibody response primarily towards the human CD52.

To collect splenocytes from the human CD52 transgenic mice, the micewere euthanized, spleens were harvested and single cell suspensions wereprepared by passing through a syringe. CD1 mice were then immunized withthe collected human CD52 positive spleen cells at 5×10⁶ in 100 μl permouse with or without Freund's Complete Adjuvant by intraperitoneal(i.p.) injection. Mice were given two booster doses every two weeksafter first immunization with transgenic mouse human CD52 positivespleen cells at 5×10⁶ in 100 μl per mouse with Freund's IncompleteAdjuvant, ip.

Eye bleeds were collected 100-200 μl per mouse in yellow-capped serumseparator tubes from all mice before immunization to determine baselevel reactivity, and a week after every round of immunization todetermine base level reactivity, and a week after every round ofimmunization to determine anti-human CD52 specific immune response. Micethat mounted high levels of anti-human CD52 reactivity as measured byFACS on CHO K1 cells engineered to express human CD52 protein, but noton parental CHO K1 cells were sacrificed, blood was harvested andspleens were collected under sterile conditions to generate hybridomas.Hybridomas were generated by using a non-secreting mouse myelomacell-line SP2/0 Ag14 or NS1 myeloma cells as fusion partners 3-4 dayspost immunization. Fused cells were placed in complete growth mediumcontaining hypoxanthine, aminopterin and thymidine to generatehybridomas. After screening many hybridoma supernatants, several cloneswere selected that produced specific anti-human CD52 antibodies and werefurther subcloned to derive a clonal population. Hybridoma clones thatproduced anti-human CD52 antibodies were scaled up for furtherdevelopment.

Example 2 PCR Analysis of Heavy and Light Chains of Mouse Anti-HumanCD52 Antibodies

A number of mouse anti-human CD52 monoclonal antibodies (FIG. 1B) wereidentified by testing hybridoma supernatants for the presence ofanti-human CD52 reactivity. Individual clones were selected and themouse heavy and light chain variable sequences were identified by PCRcloning and sequencing. The sequences of the light chains are shown inFIG. 2 as compared to YTH 34.5 HL (i.e., Campath IG Kappa (rat) and areagent antibody CF1D12 (CF1D12 Kappa) (Invitrogen Life ScienceTechnologies). Similarly, the sequences of the heavy chains are shown inFIG. 3 as compared to YTH 34.5 HL and a reagent antibody CF1D12.

A total of 10 Zunique light chain variable sequences and 11 unique heavychain variable sequences were identified. If one includes Campath® andCF1D12, 7 unique CDR-1 regions (Table 1), 8 unique CDR-2 regions (Table2) and 7 unique CDR-3 regions (Table 3) were identified within the lightchains of anti-human CD52 antibodies.

TABLE 1 Light Chain CDR-1 Sequences Light Chain CDR-1 Sequence AKASQNIDKYLN (SEQ ID NO: 27) B KSSQSLLESDGRTYLN (SEQ ID NO: 28) CKSSQSLLDSDGKTYLN (SEQ ID NO: 29) D KSSQSLLDSDGRTYLN (SEQ ID NO: 30) EKSSQSLLYSNGKTYLN (SEQ ID NO: 31) F RSSQSLVHTNGNSYLH (SEQ ID NO: 32) GRSSQSLVHTNGNTYLH (SEQ ID NO: 33)

TABLE 2 Light Chain CDR-2 Sequences Light Chain CDR-2 Sequence A NTNNLQT(SEQ ID NO: 34) B LVSNLDS (SEQ ID NO: 35) C LVSKLDS (SEQ ID NO: 36) DLVSNLGS (SEQ ID NO: 37) E LVSALDS (SEQ ID NO: 38) F LVSNLNS(SEQ ID NO: 39) G LVSHLDS (SEQ ID NO: 40) H MVSNRFS (SEQ ID NO: 41)

TABLE 3 Light Chain CDR-3 Sequences Light Chain CDR-3 Sequence ALQHISRPRT (SEQ ID NO: 42) B WQGTHFPWT (SEQ ID NO: 43) C VQGSHFHT(SEQ ID NO: 44) D VQGTRFHT (SEQ ID NO: 45) E VQGTHLHT (SEQ ID NO: 46) FSQSTHVPFT (SEQ ID NO: 47) G SQSAHVPPLT (SEQ ID NO: 48)

If one includes Campath® and CF1D12, a total of 8 unique CDR-1 regions(Table 4), 10 unique CDR-2 regions (Table 5) and 8 unique CDR-3 regions(Table 6) have been identified within the heavy chains of anti-humanCD52 antibodies.

TABLE 4 Heavy Chain CDR-1 Sequences Heavy Chain CDR-1 Sequence AGFTFTDFYMN (SEQ ID NO: 49) B GFTFSDAWMD (SEQ ID NO: 50) C RFTFSDAWMD(SEQ ID NO: 51) D GLTFSDAWMD (SEQ ID NO: 52) E GFPFSNYWMN(SEQ ID NO: 53) F GFTFNKYWMN (SEQ ID NO: 54) G GFTFNTYWMN(SEQ ID NO: 55) H GFTFTDYYMS (SEQ ID NO: 56)

TABLE 5 Heavy Chain CDR-2 Sequences Heavy Chain CDR-2 Sequence AFIRDKAKGYTTEYNPSVKG (SEQ ID NO: 57) B EIRNKAKNHVAYYAESVKG(SEQ ID NO: 58) C EIRNKANNHATYYAESVKG (SEQ ID NO: 59) DEIRNKAKNHVKYYAESVKG (SEQ ID NO: 60) E EIRNKAKNHATYYAESVKG(SEQ ID NO: 61) F EIRKKVNNHATYYAESVKG (SEQ ID NO: 62) GQIRLKSNNYATHYAESVKG (SEQ ID NO: 63) H QIRLKSDNYATHYAESVKG(SEQ ID NO: 64) I FIRNKANGYTTEYNASVKG (SEQ ID NO: 65) JFIRNKANGYTTEYSASVKG (SEQ ID NO: 66)

TABLE 6 Heavy Chain CDR-3 Sequences Heavy Chain CDR-3 Sequence AAREGHTAAPFDY (SEQ ID NO: 67) B TTLDS (SEQ ID NO: 68) C TSLDY(SEQ ID NO: 69) D TGLDY (SEQ ID NO; 70) E TPIDY (SEQ ID NO: 71) F TPVDF(SEQ ID NO: 72) G TRYIFFDY (SEQ ID NO: 73) H TRYIWFDY (SEQ ID NO: 74)

The association of specific light and heavy chain CDR regions within 13different anti-human CD52 antibodies is depicted in Table 7.

TABLE 7 Classification of Anti-Human CD52 Antibodies on the Basis of CDRComposition Clone Heavy Chain CDR-1 Light Chain Name CDR-1 CDR-2 CDR-3CDR-1 CDR-2 CDR-3 Campath A A A A A A CF1D12 B B B B B B 8G3.25.3.5 C CC C C B GMA 4G7.F3 B D C C D B GMA 9D9.A2 B E B C E B GMA B E C C C B11C11.C5 GMA 3G7.E9 B F C C F B 5F7.1.1.4 D E D D G B 12G6.15.1.2 E G EE C C 23E6.2.2.1 F H E E C D 2C3.3.8.1 G G F E C E 7F11.1.9.7 H I G F HF 4B10.1.2.4 H J H G H G Clones 8G3.25.3.5, 4G7.F3, 9D9.A2, 11C11.C5,3G7.E9, 5F7.1.1.4, 12G6.15.1.2, 23E6.2.2.1, 2C3.3.8.1, 7F11.1.9.7 and4B10.1.2.4 are hereafter referred to as 8G3, 4G7, 9D9, 11C11, 3G7, 5F7,12G6, 23E6, 2C3, 7F11 and 4B10, respectively.

TABLE 7.1 SEQ ID NOs of the CDRs of the Anti-Human CD52 Antibodies CloneHeavy Chain Light Chain Name CDR-1 CDR-2 CDR-3 CDR-1 CDR-2 CDR-3 Campath49 57 67 27 34 42 CF1D12 50 58 68 28 35 43 8G3 51 59 69 29 36 43 4G7 5060 69 29 37 43 9D9 50 61 68 29 38 43 11C11 50 61 69 29 36 43 3G7 50 6269 29 39 43 5F7 52 61 70 30 40 43 12G6 53 63 71 31 36 44 23E6 54 64 7131 36 45 2C3 55 63 72 31 36 46 7F11 56 65 73 32 41 47 4B10 56 66 74 3341 48

Example 3 Cloning of Mouse IgG Variable Region Genes from MouseHybridoma Cells to Generate a Mouse/Human Chimeric IgG1 Antibody

Actively proliferating and antibody secreting hybridoma cells were usedto isolate RNA using Trizol reagent (Gibco/BRL) following themanufacturer's suggested protocol. RNA was quantified by measuring ODusing Nanodrop, and the integrity of the RNA was determined by runningit on a gel or by using a bioanalyzer. Total RNA was reverse transcribedto cDNA and the variable regions for the heavy and light chains wereamplified by polymerase chain reaction (PCR). The cDNA was generatedusing BD Sprint PowerScript Reverse Transcriptase (Clontech) andOligo(dT) primer at 0.5 μg/μl (Invitrogen Cat# Y01212) and reverseprimers (located in the constant region of the heavy and light chains)listed numerically below at 10 μM following the manufacturer's protocol.Specifically, primers numbered 3 (SEQ ID NO: 77), 11 (SEQ ID NO: 85), 19(SEQ ID NO: 93), 20 (SEQ ID NO: 94) and 21 (SEQ ID NO: 95) wereemployed. PCR amplification of the heavy and light chain variableregions was carried out using cDNA generated as described above. 1 μl ofcDNA was mixed with forward primer and reverse primers at 10 μM each forboth heavy and light chains and mixed with PCR super mix (Invitrogen) inthe presence of 2 μl of MgCl₂ at 25 mM. The PCR program was run in thefollowing steps: 1) 95° C. for 2 minutes; 2) 95° C. for 30 seconds; 3)56° C. for 30 seconds; 4) 68° C. for 45 seconds; 5) Repeat steps 2 to 425 times; 6) 68° C. for 10 minutes and hold at 16° C. The PCR productwas analyzed on a 2% gel for the presence of variable region sequenceproduct of about 300-400 bp in size and the appropriate bands werecloned into pCR2.1-TOPO TA cloning Kit (Invitrogen) following themanufacturer's instructions and the cloned and sequence confirmed usingM13 primers. Primers used for reverse transcribing and for PCRamplification of light chain and heavy chain sequences are provided:

Light chain primers (SEQ ID NO: 75)  1) Lead-ML kappa = 5′ATGGGCWTCAARATGRARWCWCAT 3′ (Forward primer in leader sequence)(SEQ ID NO: 76)  2) FR1-ML kappa = 5′ GAYATTGTGMTRACMCARKMTCAA 3′(Forward primer in the frame work 1) (SEQ ID NO: 77) 3) ML kappa const = 5′ ACTGGATGGTGGGAAGATGGA 3′(Reverse primer in constant region) (SEQ ID NO: 78)  4) VK-MK = 5′GAYATTGTGMTSACMCARWCTMCA 3′ (Forward primer in the frame work 1)(SEQ ID NO: 79)  5) MKC-Const = 5′ GGATACAGTTGGTGCAGCATC 3′(Reverse primer in constant region) Heavy chain primers (SEQ ID NO: 80) 6) MH-SP-ALT1 = 5′ ATGRASTTSKGGYTMARCTKGRTT 3′(Forward primer in leader sequence) (SEQ ID NO: 81)  7) MH-SP-ALT2 = 5′ATGRAATGSASCTGGGTYWTYCTCT 3′ (Forward primer in leader sequence)(SEQ ID NO: 82)  8) MH-FR1 = 5′ SAGGTSMARCTGCAGSAGTCT 3′(Forward primer in the frame work 1) (SEQ ID NO: 83)  9) MH-FR1-1 = 5′SAGGTGMAGCTCSWRSARYCSGGG 3′ (Forward primer in the frame work 1)(SEQ ID NO: 84) 10) MH-J2 = 5′ TGAGGAGACTGTGAGAGTGGTGCC 3′(Reverse primer in J region) (SEQ ID NO: 85) 11) MH-gamma-const = 5′AYCTCCACACACAGGRRC CAGTGGATAGAC 3′ (Reverse primer in constant region)(SEQ ID NO: 86) 12) VH MH1 = 5′ SARGTNMAGCTGSAGSAGTC 3′(Forward primer in the frame work 1) (SEQ ID NO: 87) 13) VH MH2 = 5′SARGTNMAGCTGSAGSAGTCWGG 3′ (Forward primer in the frame work 1)(SEQ ID NO: 88) 14) VH MH3 = 5′ CAGGTTACTCTGAAAGWGTSTG 3′(Forward primer in the frame work 1) (SEQ ID NO: 89) 15) VH MH4 = 5′GAGGTCCARCTGCAACARTC 3′ (Forward primer in the frame work 1)(SEQ ID NO: 90) 16) VH MH5 = 5′ CAGGTCCAACTVCAGCARCC 3′(Forward primer in the frame work 1) (SEQ ID NO: 91) 17) VH MH6 = 5′GAGGTGAASSTGGTGGAATC 3′ (Forward primer in the frame work 1)(SEQ ID NO: 92) 18) VH MH7 = 5′ GATGTGAACTTGGAAGTGTC 3′(Forward primer in the frame work 1) (SEQ ID NO: 93) 19) IgG1 = 5′ATAGACAGATGGGGGTGTCGTTTTGGC 3′(Reverse primer in mouse IgG1 CH1 constant  region) (SEQ ID NO: 94)20) IgG2A = 5′ CTTGACCAGGCATCCTAGAGTCA 3′(Reverse primer in mouse IgG2A CH1 constant  region) (SEQ ID NO: 95)21) IgG2B = 5′ AGGGGCCAGTGGATAGAGTGATGG 3′(Reverse primer in mouse IgG2B CH1 constant  region)

Degenerate primers led to some degeneracy in the 5′ end of the framework 1 region of both heavy and light chains. The consensus DNA sequencefrom several independent heavy chain variable region clones and fromlight chain variable region clones was used to derive the amino acidsequence.

Functional chimeric anti-CD52 antibodies were produced by joining theheavy chain and light chain variable regions to the DNA encoding humanIgG1 heavy chain (identical sequence to that found in Campath-1H®) andhuman kappa light chain constant region (identical sequence to thatfound in Campath-1H®), respectively. To generate pCEP4 (Invitrogen)light chain vector encoding CD52 antibody light chain, the light chainvariable sequence was PCR amplified and engineered by ligase independentcloning into the pCEP4 LIC light chain vector to have the human kappasignal sequence in the 5′ end and the light chain constant region in the3′ end. Similarly, to generate pCEP4 heavy chain vector, the variableregion of the heavy chain sequence was engineered by ligase independentcloning into the pCEP4 LIC heavy chain vector to have the human kappachain signal sequence in the 5′ end and the heavy chain constant regionencompassing CH1, hinge, CH2 and CH3 regions in the 3′ end. The constantregion amino acid sequences for both heavy and light chains areidentical to that of the constant regions present in Campath1H antibody.

Briefly, pCEP4 LIC vector was digested with BfuA1 (New EnglandBiolabs-NEB) in appropriate buffer following the manufacturer'srecommendations and after complete digestion, the vector was purifiedusing PureLink PCR Purification Kit (Invitrogen). The linearized plasmidwas then treated with T4 DNA polymerase (New England Biolabs) togenerate single-stranded ends and was used to clone the variable regionfragment. Heavy chain specific pCEP4 LIC vector was used for cloningheavy chain variable region and light chain specific pCEP4 LIC vectorwas used for cloning light chain variable region. Variable region insertwas generated by PCR using pCR2.1-TOPO heavy chain variable regioncontaining plasmid or pCR2.1-TOPO light chain variable region containingplasmid as template and primers that contain variable chain specificsequence and vector overhangs. VENT DNA polymerase (New England Biolabs)was used for PCR amplification of the insert. PCR-amplified insert wasgel purified and treated with T4 DNA polymerase to generatesingle-stranded ends. Prepared vectors for heavy chain and light chainand respective variable region insert fragments were combined andincubated at room temperature for 10 minutes and used to transformTOPO10 cells (Invitrogen), ampicillin resistant colonies were picked andsequence verified. pCEP4 heavy chain and pCEP4 light chain clones thathad the correct heavy chain and light chain sequences inserted in-framewere amplified and used for protein production. The heavy chainconstruct was co-transfected with the corresponding light chainconstruct in a 1:1 ratio into HEK293 cells (Invitrogen) using thecationic lipid Lipofectamine™ 2000 (Invitrogen). The conditioned mediumwas harvested three days after the transfection and the chimericantibody was purified using protein A chromatography. For thischromatography method, the medium was added to protein A and washed with50 column volumes of PBS. The chimeric antibody was eluted with 5 columnvolumes of 12.5 mM citric acid, pH 3.0. The pH of the eluted antibodywas neutralized by addition of 0.5 M HEPES. The buffer was exchangedinto PBS by using a PD-10 gel filtration column.

Example 4 Analysis of the Epitope Specificities of Chimeric Anti-HumanCD52 Monoclonal Antibodies

The epitope specificities of the clones were determined by assessing theability of the chimeric antibodies to bind to a panel of cell linesengineered to express mutants of human CD52 (FIG. 4) generated byalanine scanning mutagenesis. Antibody substitution of the first 10amino acids of the 12 amino acid extracellular region of CD52 wasconducted on human CD52 cDNA in pcDNA3.1 expression vector (Invitrogen)using the STRATAGENE QUIKCHANGE II XL site-directed mutagenesis kit.pcDNA3.1 vector encoding wild type or mutant CD52 sequence wassequence-verified and transfected into CHO cells using Lipofectamine™and by selecting in media containing G418 to generate CHO cell linesthat expressed wild type or alanine mutant CD52. Epitope specificbinding of anti-human CD52 chimeric antibodies was determined bymeasuring the binding of the antibodies against the wild type and mutantCD52 expressing cells by FACS. FACS analysis was carried out bydetecting the binding of chimeric anti-CD52 antibodies usingPE-conjugated goat anti-human secondary antibody (Jackson ImmunoResearchLabs). FIGS. 5A-5C show the Mean Fluorescence Intensity (MFI) ofanti-CD52 monoclonal antibodies to wild type and mutant CD52 expressingcell lines. Even though CD52 is a very short, 12 amino acid, GPIanchored protein, the FACS results clearly define that there are threesets of antibodies: (1) N-terminal binding group (such as 4B10); (2)middle binding group (such as 4G7, 9D9 and 11C1) and (3) C-terminalbinding group (such as 23E6, 12G6, and 2C3). The epitope specificitiesof the anti-human CD52 monoclonal antibodies (identified by theabbreviated names described at the end of Example 2) are summarized inTable 8.

TABLE 8 Characteristics of 11 Mouse Anti- Human CD52 MonoclonalAntibodies Epitope Clone Isotype Specificity Rat YTH34.5HL IgG2a9-10-11-12 Mouse CF1D12 IgG3 3-4-5-6-7 8G3.25.3.5 IgG3 Not confirmed4G7.F3 IgG3 3-4-5-6-7 9D9.A2 IgG3 3-4-5-6-7 11C11.C5 IgG3 1-3-4-5-6-73G7.E9 IgG2b 1-3-4-5-6-7 5F7.1.1.4 IgG3 1-3-4-5-6-7-10 12G6.15.1.2 IgG37-8-9 23E6.2.2.1 IgG3 7-8-9 2C3.3.8.1 IgG3 7-8-9-10 7F11.1.9.7 IgG11-2-3-4-5 4B10.1.2.4 IgG2a 1-2-3-4-5

CD52 is an extremely small antigen but possesses a relatively large,hydrophilic N-linked glycan moiety as well as a hydrophobic GPI-anchor.To explore the possibility that the sugars might constitute all or partof an epitope recognized by the anti-CD52 antibodies, samples ofaffinity purified CD52 from CHO-CD52 cells were treated with theendoglycosidase, PNGase-F, to completely remove N-linked sugars from theantigen. Treated and mock-treated control samples were then resolved bySDS-PAGE, blotted to polyvinylidene fluoride (PVDF) membrane(Invitrogen), probed with 3 μg/ml final of each of the anti-CD52chimeric monoclonal antibodies indicated, and subsequently developedaccording to standard western blotting procedures using enhancedchemiluminescent detection. Blots with Campath-1H® (C1H) and withsecondary antibody alone (2° Alone) were run as positive and negativecontrols, respectively, and probed with each of the monoclonalantibodies (FIG. 5D). The results revealed different binding preferencesamongst the antibodies for glycosylated versus de-glycosylated CD52.This characterization allowed for the categorization of the elevenantibodies into four types of binding groups:

-   -   1. Antibodies exhibiting binding with no apparent preference for        glycosylated versus de-glycosylated CD52 (4G7, 9D9)    -   2. Antibodies exhibiting binding specific for glycosylated CD52        (7F11, 4B10)    -   3. Antibodies exhibiting binding specific for de-glycosylated        CD52 (8G3)    -   4. Antibodies exhibiting binding preferential for        de-glycosylated over glycosylated CD52 (12G6, 5F7, 23E6, 2C3,        11C11, 3G7)

Example 5 CDC Activity of Chimeric Anti-CD52 Antibodies

A complement-dependent cytotoxicity (CDC) assay was performed asdescribed below. Briefly, CHO K1 cells engineered to express CD52protein (CHO-CD52) were used as target cells and labeled with Na₂ ⁵¹CrO₄(New England Nuclear, Boston, Mass.) at 37° C. for 1-2 hrs. The cellswere washed, resuspended with X-Vivo media, and mixed with anti-humanCD52 antibodies to final concentration of 2.2 μg/ml. Human complement(Sigma) was added to the experimental wells to a final concentration of10%. After a 1-5-hour incubation, 25 μl of cell-free supernatant wascollected from each well and counted in a MICROBETA TRILUX ScintillationCounter (Wallac, Gaithersburg, Md.). The amount of ⁵¹Cr spontaneouslyreleased was obtained by incubating target cells in medium alone.Spontaneous release from target cells was typically less than 20%. Thetotal amount of ⁵¹Cr incorporated was determined by adding 1% TritonX-100 in distilled water, and the percentage lysis was calculated asfollows: [(sample counts per minute (c.p.m.)−spontaneous c.p.m.)/(totalc.p.m.−spontaneous c.p.m.)]×100.

Twelve different chimeric anti-CD52 antibodies (mouse variable regionand human IgG1 constant region) were tested in CDC assay with humancomplement on CHO-CD52 cells. Campath-1H® humanized antibody was used asa positive control. A negative control was Campath-1H® null (anon-cell-binding minimal mutant of Campath-1H®—two point mutations in H2loop-heavy chain CDR2 region (K52bD and K53D; Gilliland L K et al.,Journal of Immunology, 162:3663-3671 (1999)). The results indicate thatthe chimeric antibodies are capable of mediating CDC killing onCD52-expressing cells. Some of the chimeric antibodies mediated robustkilling equivalent or better than Campath® (FIG. 6).

Example 6 ADCC Activity of Chimeric Anti-CD52 Antibodies

An antibody-dependent cytotoxicity (ADCC) assay was performed asdescribed below. Briefly, CHO K1 cells engineered to express CD52protein (CHO-CD52) were used as target cells and labeled with Na₂ ⁵¹CrO₄(New England Nuclear, Boston, Mass.) at 37° C. for 1-2 hrs. The cellswere washed, resuspended with X-Vivo media, and mixed with anti-humanCD52 antibodies to final concentration of 1.1 μg/ml. Human PBMC wereused as effectors cells and were added at a 1:100 target-to-effectorcell ratio. After a 6 hr-overnight incubation, 25 μl of cell-freesupernatant was collected from each well and counted in a MICROBETATRILUX Scintillation Counter (Wallac, Gaithersburg, Md.). The amount of⁵¹Cr spontaneously released was obtained by incubating target cells inmedium alone. Spontaneous release from target cells was typically lessthan 20%. The total amount of ⁵¹Cr incorporated was determined by adding1% Triton X-100 in distilled water, and the percentage lysis wascalculated as follows: [(sample c.p.m.−spontaneous c.p.m.)/(totalc.p.m.−spontaneous c.p.m.)]×100.

Twelve different chimeric anti-CD52 antibodies (mouse variable regionand human IgG1 constant region) were tested in ADCC assay using humanPBMC as effector cells. Campath-1H® humanized antibody was used as apositive control. Used as a negative control was Campath-1H® null (anon-cell-binding minimal mutant of Campath-1H®—two point mutations in H2loop-heavy chain CDR2 region (K52bD and K53D; Gilliland, 1999, supra).The results indicate that the chimeric antibodies are capable ofmediating ADCC killing on CD52-expressing cells. Some of the chimericantibodies mediated robust killing equivalent or better than Campath-1H®(FIG. 7).

Example 7 Evaluation of the Binding of Chimeric Anti-CD52 Antibodies toDefined Lymphocyte Population

The following fluorochrome conjugated antibodies were used for flowcytometric analysis: anti-CD3-FITC, anti-CD27-PE, anti-CD62L-PE Cy5,anti-CD56-PE Cy7, anti-CD16-APC Cy7 (BD Biosciences, San Diego, Calif.),anti-CD45RA-ECD (Beckman Coulter), anti-CD19-Pacific Blue, anti-CD4-APCCy5.5 and anti-CD8 pacific orange (Invitrogen, CA). All the mousechimeric anti-human CD52 antibodies as well as the humanized Campath-1H®were conjugated to Alexa fluor 647 (BD Pharmingen). Healthy humanperipheral blood mononuclear cells were obtained either fromcryopreserved buffy coats or from mononuclear cells separated from bloodof normal donors obtained from commercial vendors (Bioreclamation, NY,USA). For enrichment of mononuclear cells, human peripheral blood wasdiluted 1:1 with sterile phosphate buffered saline (PBS) and carefullylayered over Ficoll-hypaque (GE Healthcare Bio-Sciences, Uppsala,Sweden) and centrifuged for 30 min at room temperature. The interphaselayer of mononuclear cells was drawn out and washed in PBS containing 5%fetal bovine serum (FACS buffer). Contaminating red blood cells (RBCs)were lysed with RBC lysing solution (Sigma, St. Louis, Mo., USA). Cellswere resuspended in cold FACS buffer and the debris was removed using a40 micron filter. Ten color flow cytometry was performed to evaluate thebinding ability of 9 chimeric anti-human CD52 antibodies (4B10, 7F11,9D9, 5F7, 2C3, 4G7, 23E6, 8G3, 3G7) as compared to Campath-1H®.

Briefly, replicates of 1×10⁶ PBMC's in FACS buffer were incubated with acocktail of pre-titrated dilutions of antibodies against CD3, CD27,CD45RA, CD62L, CD56, CD19, CD8, CD4, CD16 along with one of 9 chimericanti-human CD52 antibodies (4B10, 7F11, 9D9, 5F7, 2C3, 4G7, 23E6, 8G3,3G7) for 30 min at 4° C. Cells were washed and fixed in PBS containing1% paraformaldehyde. 100,000 events of the stained cells were acquiredon BD LSR-II (BD Biosciences, San Jose, Calif.) and the data wasanalyzed using FlowJo 7.2 version Software (Tree Star, Inc, Oregon,USA). Multiple subsets with distinct phenotypic characteristics havebeen defined among B and T lymphocytes and CD52 has been shown to beexpressed on all human lymphocytes. Ten color flow cytometry analysiswas performed to identify the lymphocyte subsets, and to assesssimilarities and the differences in the binding characteristics ofanti-CD52 antibodies to cell surface CD52 on defined subsets. Using acombination of markers, 11 phenotypically distinct cell populationscorresponding to B, T and NK cell lineages were first defined from thelymphocyte gate. The intensity of staining which corresponds to theability of anti-CD52 antibodies to detect CD52 expression was thenassessed. The histograms (FIGS. 8A-8C) show a comparison of the level ofdetection of CD52 with each antibody on individual lymphocytepopulations. The data shows that the antibodies exhibit significantdifferences in binding to CD52. The level of detection with 4B10, 9D9,7F11 and Campath-1H® are comparable, although 4B10 consistently showsthe highest level of detection than other antibodies includingCampath-1H®, on almost all the cell subsets examined. On the other hand,the detection level of CD52 with 3G7, 4G7, 8G3 and 23E6 antibodies issignificantly lower. The results indicate a hierarchy within theantibodies with respect to their ability to recognize CD52 on differentcell populations with 4B10 being highest and 3G7 being the lowest.Interestingly, these differences are less obvious on CD4 effector andmore so on NK cell subsets on which CD52 appears to be expressed atrelatively lower levels. The variations in the binding characteristicsindicate that the properties of the chimeric antibodies not only differsignificantly from Campath-1H® but also reflect differences inproperties among the antibodies.

Example 8 Analysis of Chimeric Anti-CD52 Antibodies in Human CD52Transgenic Mice (7F11, 8G3, 23E6, 12G6, 4B10 and 5F7)

Human CD52 transgenic mice were administered either Campath® or chimericanti-CD52 antibodies (7F11, 8G3, 23E6, 12G6, 4B10 and 5F7) to examinethe level of lymphocyte depletion. Mice were injected intra-peritoneallywith either Campath® or the chimeric anti-CD52 antibodies in a 100 μlvolume at a dose of 1 mg/kg. Three days later mice were sacrificed andblood and spleens were collected to determine the level of B and T-celldepletion. Flow cytometry was utilized to evaluate the absolute numbersof total T helper cells, cytotoxic T cells, and B cells present in thecirculating peripheral blood or spleens of huCD52 transgenic mice. Theselymphocyte populations were defined by their surface expression of thefollowing protein antigens: CD4 expression identifies the T helper cellpopulation, CD8 expression identifies the cytotoxic T cell populationand CD19 expression identifies all mature B cell populations. Asignificant level of T and B-cell depletion was observed for both the12G6 and 4B10 antibodies, which was comparable to the depletion observedwith Campath®. Treatment with either Campath®, the chimeric 12G6 or thechimeric 4B10 antibody significantly reduced T and B cells in both theblood and spleens of treated mice at this dose level. The 7F11 and 5F7chimeric antibodies resulted in significant levels of T cell depletionlevel in the blood and spleen but were less effective at depleting Bcells in both compartments. Treatment with the 23E6 antibody resulted ina moderate level of depletion at this dose while little to no depletionwas observed with the lower affinity 8G3 antibody.

FIGS. 9A-9C show the level of CD4 T cells, CD8 T cells and CD19 B cellsin the blood 72 hours after dosing with the chimeric antibodies. FIGS.10A-10C show the level of CD4 T cells, CD8 T cells and CD19 B cells inthe spleen 72 hours after dosing.

Example 9 Analysis of Chimeric Anti-CD52 Antibodies in Human CD52Transgenic Mice (2C3, 3G7, 4B10, 9D9, and 11C11)

Human CD52 transgenic mice were administered either Campath® or chimericanti-CD52 antibodies (2C3, 3G7, 4B10, 9D9 and 11C11) to examine thelevel of lymphocyte depletion. Mice were injected intravenously witheither Campath® or the chimeric anti-CD52 antibodies in a 100 μl volumeat a dose of 1 mg/kg. Three days later mice were sacrificed and bloodand spleens were collected to determine the level of B and T-celldepletion. Flow cytometry was utilized to evaluate the absolute numbersof total T helper cells, cytotoxic T cells, and B cells present in thecirculating peripheral blood of huCD52 transgenic mice. These lymphocytepopulations were defined by their surface expression of the followingprotein antigens: CD4 expression identifies the T helper cellpopulation, CD8 expression identifies the cytotoxic T cell populationand CD19 expression identifies all mature B cell populations. Asignificant level of T and B cell depletion was observed for severalantibodies in both the blood and spleen. The depleting activity for 2C3and 9D9 was comparable to that observed with Campath® with significantlevels of CD4 and CD8 T cells and CD19 B cells being depleted. Treatmentwith chimeric 4B10 also resulted in a significant decrease in thenumbers of lymphocytes in the blood of transgenic mice. While treatmentwith either the chimeric antibody 3G7 or 11C11 antibody significantlydepleted T cells in the blood, the level of B cells present were notsignificantly affected at this dose.

FIGS. 11A-11C show the level of CD4 T cells, CD8 T cells and CD19 Bcells in the blood 72 hours after dosing with the chimeric antibodies.

Example 10 Analysis of the Efficacy of Anti-CD52 Antibodies (7F11, 4B10and 12G6)

Forty SCID mice (n=8 per group) were injected with 1×10⁶ B104 tumorcells in 100 μl volume on the right flank. On day 11 post tumor cellinjection, treatment began with Campath®, 7F11, 4B10 or 12G6 chimericantibodies. Antibodies were administered once weekly at 10 mg/kg byintraperitoneal injection throughout the remainder of the experiment.All mice in the untreated group developed progressively growing tumorsrequiring sacrifice with a median survival of 29 days. Treatment withCampath® resulted in a statistically significant increase in survivalcompared to the untreated group (median survival (MS) of 50 days andp<0.0001). Treatment with the chimeric anti-CD52 antibodies alsoresulted in a statistically significant increase in survival compared tountreated mice (p<0.0001 for 7F11 and 4B10 and p=0.0020 for 12G6). Basedon survival rates, the activity of both 7F11 and 4B10 antibodies appearsto be greater than Campath® (63% survival for 7F11 and 75% survival for4B10 compared to 50% survival for Campath®). FIG. 12 shows the percentsurvival of the mice after treatment.

Example 11 Analysis of the Efficacy of Anti-CD52 Antibodies (2C3, 8G3and 23E6)

Forty SCID mice (n=8 per group) were injected with 1×10⁶ B104 tumorcells in a 100 μl volume on the right flank. On day 11 post tumor cellinjection, treatment began with either Campath®, 2C3, 8G3 or 23E6chimeric antibodies. Antibodies were administered once weekly at 10mg/kg by intraperitoneal injection throughout the remainder of theexperiment. All mice in the untreated group developed progressivelygrowing tumors requiring sacrifice with a median survival of 26 days.Treatment with Campath®, 23E6, and the 2C3 antibody resulted instatistically significant increases in survival (p=0.0025, p=0.0007, andp=0.0002 respectively).

FIG. 13 shows the percent survival of the mice after treatment.

Example 12 Analysis of the Efficacy of Chimeric Anti-CD52 Antibodies ina Xenograft Tumor Model (9D9 and 4B10)

Forty SCID mice (n=8 per group) were injected with 1×10⁶ B104 tumorcells in a 100 μl volume on the right flank. On day 11 post tumor cellinjection, treatment began with either Campath®, 9D9 or 4B10 chimericantibody. Antibodies were administered once weekly at 10 mg/kg byintraperitoneal injection throughout the remainder of the experiment.All mice in the untreated group developed progressively growing tumorsrequiring sacrifice with a median survival of 27 days. Treatment withCampath® resulted in a statistically significant increase in survivalcompared to the untreated group (median survival not achieved andp<0.0001). Treatment with the chimeric anti-CD52 antibodies alsoresulted in a statistically significant increase in survival compared tountreated mice (p<0.0001 for 9D9 and 4B10). Statistical analysis of thesurvival curves reveals that the 9D9 chimeric antibody displayedactivity comparable to Campath® (p=0.0675) in this experiment. FIG. 14shows the percent survival of the mice after treatment.

Example 13 Analysis of the Efficacy of Chimeric Anti-CD52 Antibodies ina Xenograft Tumor Model (2C3 and 11C11)

Forty SCID mice (n=8 per group) were injected with 1×10⁶ B104 tumorcells in a 100 μl volume on the right flank. On day 11 post tumor cellinjection, treatment began with either Campath®, 2C3 or 11C11 chimericantibody. Antibodies were administered once weekly at 10 mg/kg byintraperitoneal injection throughout the remainder of the experiment.All mice in the untreated group developed progressively growing tumorsrequiring sacrifice with a median survival of 32 days. Treatment withCampath® resulted in a statistically significant increase in survivalcompared to the untreated group (median survival not achieved andp<0.0001). Treatment with the chimeric anti-CD52 antibodies alsoresulted in a statistically significant increase in survival compared tountreated mice (p<0.0001 for 2C3 and p=0.0004 for 11C11). Statisticalanalysis of the survival curves reveals that both the 2C3 and 11C11chimeric antibodies displayed activity comparable to Campath® (p=0.3173for 2C3 and p=0.9703 for 11C11). FIG. 15 shows the percent survival ofthe mice after treatment with Campath®, 2C3 chimeric antibody or 11C11chimeric antibody.

Example 14 Generation and Analysis of Humanized Anti-CD52 Antibody 4B10

Humanized anti-human CD52 antibody 4B10 was generated by grafting theCDR regions from the mouse 4B10 antibody into a human antibody variableregion framework. Mouse 4B10 heavy chain and light chain sequences wereevaluated by a web-based sequence alignment in order to identify a humangermline heavy chain and light chain framework sequence that would serveas a suitable acceptor for the CDR graft (FIG. 16). The residuesdefining the CDR regions by Kabat and IMGT® were superimposed into humanframework regions that have high sequence identity to generate humanizedheavy chain and light chain sequences. Visual inspection and sequenceanalysis of the superimposed 4B10 heavy and light chain sequences wascarried out to identify the most suitable acceptor sequence. Of all thegermline sequences that have high similarity, the VH3-72 germlinesequence for heavy chain and the VK2-A18b for light chain (human germline sequences can be found at the website described in the publicationby Tomlinson, I M, et al., EMBO J., 14(18):4628-4638 (1995); Cook, G P.,et al., Nature Genetics, 7:162-168 (1994)) were selected from their highdegree of homology, sequence similarity to mouse framework regions andfor minimal disruption of CDR loop structure as CDR acceptor sequence.CDR1, 2, and 3 sequences of heavy chain and light chain for 4B10 weregrafted into VH3-72 and VK2-A18b human framework regions respectively togenerate humanized heavy chain and light chain sequences for 4B10(illustrated in FIG. 17; FIG. 110).

Example 15 Assessment of the Binding Activities of Chimeric andHumanized 4B10 Monoclonal Antibodies

Chimeric and humanized 4B10 antibodies were produced and purified asdescribed in Example 3 and analyzed for their ability to bind to the Bcell line B104, which endogenously expresses CD52, by FACS. Briefly,2×10⁵ B104 cells were incubated with antibody (0.02 μg/ml to 16.7 μg/ml)in PBS containing 5% fetal bovine serum and 5% goat serum. The boundantibody was detected with FITC labeled goat anti-human secondaryantibody which detected chimeric or humanized anti-CD52 antibodies.Labeled cells were analyzed using a FACSCalibur system (BectonDickinson). FIG. 18 shows the fold increase in Geometric meanfluorescence intensity of each sample normalized (divided) to that of2°-only sample. The 11 different concentrations (12^(th) point on X axisis secondary alone) of the humanized and chimeric antibody used in theassay is shown on the X axis and the Geo Mean fold increase in the meanfluorescence is on the Y axis. The results indicate that the humanized4B10 antibody bound as well or slightly better than chimeric 4B10antibody to CD52 expressing cells.

Example 16 Assessment of the ADCC Activities of Chimeric and Humanized4B10 Monoclonal Antibodies

Humanized and chimeric 4B10 antibodies were evaluated for their abilityto mediate ADCC killing of CD52-expressing cells. An ADCC assay wascarried out as described above in Example 6. Briefly, CHO K1 cellsengineered to express CD52 protein (CHO-CD52) were used as target cellsand labeled with Na₂ ⁵¹CrO₄ (New England Nuclear, Boston, Mass.) at 37°C. for 1-2 hrs. The cells were washed, resuspended in RPMI 1640 mediawith 10% FCS, and mixed with chimeric or humanized 4B10 antibodies atvarious concentrations ranging from 10 μg/ml to 0.01 μg/ml. Human PBMCwere used as effectors cells and were added at 1:50 target-to-effectorcell ratio. After a 6 hr-overnight incubation, 25 μl of cell-freesupernatant was collected from each well and counted in a MICROBETATRILUX Scintillation Counter (Wallac, Gaithersburg, Md.). The amount of⁵¹Cr spontaneously released was obtained by incubating target cells inmedium alone. Spontaneous release from target cells was typically lessthan 20%. The total amount of ⁵¹Cr incorporated was determined by adding1% Triton X-100 in distilled water, and the percentage lysis wascalculated as follows: [(sample c.p.m.−spontaneous c.p.m.)/(totalc.p.m.−spontaneous c.p.m.)]×100.

FIG. 19 illustrates the concentrations of control, chimeric andhumanized 4B10 antibodies used in the assay (X axis) and the Y axisshows % specific killing. The results indicate that humanized 4B10antibody mediated equivalent or slightly better ADCC killing thanchimeric 4B10 antibody. The control IgG1 isotype control showed only lowlevels of background killing at the concentrations tested.

Example 17 Assessment of the CDC Activities of Chimeric and Humanized4B10 Monoclonal Antibodies

Humanized and chimeric 4B10 antibodies were evaluated for their abilityto mediate cytotoxic effect on B104 cells that endogenously express CD52in the presence of human complement. CellTiter Glo kit (Promega) wasused to determine the live cells remaining in the assay. Briefly, B104cells (target cells) were plated at 2.5×10⁴ cells/well in a 96 wellplate and were mixed with chimeric or humanized 4B10 antibody at variousconcentrations ranging from 1 μg/ml to 25 μg/ml and human complement toa final concentration of 10%. Complement alone without the antibody andantibody alone without complement were used as controls to determine thebackground. After three hours of incubation at 37° C., plates werecentrifuged for 3 min at 1500 rpm and the live cells present in thepellet were determined using CellTiter Glo assay. Plates were read onEnvision machine. FIG. 20 shows the live cells present in the assay asmeasured using CellTiter Glo assay. Again, with the increasingconcentrations of the humanized and chimeric 4B10 antibody there is adecrease in the number of live cells. These results suggest that thehumanized antibody performed as well as or slightly better than chimeric4B10 antibody in CDC mediated killing of B104 cells.

Example 18 Analysis of Pharmacokinetic Profile of Chimeric And HumanizedAnti-CD52 Antibodies in CD52 Transgenic Mice (12G6, 7F11, Chimeric andHumanized 4B10)

Human CD52 transgenic mice were administered one of Campath®, 12G6,7F11, and chimeric and humanized 4B10 anti-CD52 antibodies to examinethe level of lymphocyte depletion. Mice were injected intravenously withone of those antibodies in a 100 μl volume at a dose of 1 mg/kg. Foranalysis of anti-antibody responses, 100 μl of blood was collected intoserum separator tubes via puncture of the retro-orbital plexus at 2hours, 1, 2, 4, 7, and 10 days post antibody injection. ELISA analysiswas used to determine the level of circulating human IgG1 in each serumsample. Based on circulating levels of antibody, there appears to belittle to no difference between Campath®, 7F11, and the chimeric andhumanized forms of 4B10. The 12G6 antibody displayed lower cmax valuesfollowing injection, suggesting that this antibody may be degraded morequickly. FIG. 21 shows the pharmacokinetic profile of Campath®, 12G6(chimeric), 7F11 (chimeric), 4B10 (chimeric) and 4B10 (humanized)antibodies.

Example 19 Analysis of the Depleting Activity of Chimeric and HumanizedAnti-CD52 Antibodies in CD52 Transgenic Mice (Chimeric and Humanized4B10)

Human CD52 transgenic mice were administered either Campath® or chimericor humanized 4B10 anti-human CD52 antibody to examine the level oflymphocyte depletion. Mice were injected intravenously with eitherCampath® or the chimeric or humanized 4B10 anti-human CD52 antibody in a100 μl volume at a dose of 0.1 mg/kg. Three days later mice weresacrificed and blood and spleens were collected to determine the levelof B and T-cell depletion. Flow cytometry was utilized to evaluate theabsolute numbers of total T helper cells, cytotoxic T cells, and B cellspresent in the circulating peripheral blood of the huCD52 transgenicmice. These lymphocyte populations were defined by their surfaceexpression of the following protein antigens: CD4 expression identifiesthe T helper cell population, CD8 expression identifies the cytotoxic Tcell population and CD19 expression identifies all mature B cellpopulations. Comparison of the depleting activity in the spleen revealedthat there was no difference in the level of T cells depleted followingadministration of either Campath® or the chimeric or humanized forms of4B10. Due to the low dose used, only a modest level of depletion of Bcells was observed in the spleen. On a per animal basis it appears thatthe humanized 4B10 antibody is as good or slightly better than Campath®at mediating lymphocyte depletion. FIGS. 22A-22C show the level of CD4 Tcells, CD8 T cells and CD19 B cells in the blood 72 hours after dosingwith the chimeric and humanized antibodies.

Example 20 Relative Binding Efficiency of Anti-Human CD52 Antibodies

The EC50 values of selected anti-CD52 antibodies were estimated usingCHO cells engineered to express CD52. CHO-CD52 cells were trypsinized in0.25% trypsin, collected, and rinsed with PBS/5% FBS. Cells were thendeposited into round-bottom 96 well plates at 1E5 cells per well.Primary antibody staining was done with a 12 point serial dilution (1:2)of each anti-CD52 chimeric antibody starting at 50 μg/mL.FITC-conjugated goat FAB2 fragment of anti-human Fc gamma at 10 μg/mL(Jackson 109-096-098) secondary was used. Cells were washed 3 times inice-cold PBS/5% FBS before and after each incubation. Cells were fixedwith PBS containing 2% methanol-free paraformaldehyde and evaluated byflow cytometry. The flow cytometry data was analyzed using Graph padPrizm software to determine EC50 value with 95% confidence interval.

Binding data (FIG. 23) indicates that the new CD52 antibodies not onlyhave different epitope specificities as mentioned earlier, but also havedifferent binding characteristics as shown in the table given below.Campath-1H®, 7F11, 4B10, 2C3 and 12G6 chimeric antibodies showedrelatively similar EC 50 values between 0.5 to 2.5 μg/ml/. 9D9 chimericantibody showed slightly different binding characteristics with EC50value around 5 to 7 μg/ml. 4B10 humanized antibody showed similarbinding characteristics as that of chimeric 4B10 antibody, indicatingthat the humanized antibody retained the binding characteristics as thatof chimeric 4B10 antibody.

TABLE 9 EC50 (μg/mL) Clone ID Mean STDEV C1H* 1.36 0.46 2C3-Chi 1.320.33 4B10-Chi 2.18 0.33 4B10-H1/K1 2.23 0.50 7F11-Chi 2.22 0.29 9D9-Chi6.05 1.18 12G6-Chi 0.95 0.21 *C1H refers to Campath-1H ®.

Example 21 Humanization of Anti-CD52 Antibody Clone 7F11

Humanization of anti-human CD52 antibody clone 7F11 was performed bygrafting the CDR regions from the mouse 7F11 antibody into a humanantibody variable region framework as described in Example 14 for 4B10antibody humanization. CDR-1, CDR-2, and CDR-3 sequences of the heavychain and light chain of 7F11 were grafted into VH3-72 and VK2 A18bhuman framework regions, respectively. The human JH6 (WGQGTTVTVSS: SEQID NO: 133) and JK2 (FGQGTKLEIK: SEQ ID NO: 134) sequences were selectedas the C-terminal peptides for the humanized heavy and light chains,respectively, to generate humanized heavy chain (7F11-SFD1 and7F11-SFD2) and humanized light chain (7F11-VK2) variable regionsequences for 7F11 (FIG. 24). The two humanized heavy chain variableregion sequences (7F11-SFD1 and 7F11-SFD2) differ by one amino acidresidue in the CDR-3 region. The 7F11-SFD1 version has a threonine atposition 93 (denoted by the Kabat numbering system), while the 7F11-SFD2version has an alanine at this position. Position 93 is underlined forboth 7F11-SFD1 and 7F11-SFD2 in FIG. 24.

The full-length heavy chain amino acid sequence of 7F11-SFD1 (SEQ ID NO:274) and the full-length light chain amino acid sequence of 7F11-K2 (SEQID NO: 275) are shown in FIG. 107.

Example 22 Assessment of the Binding Activities of Chimeric andHumanized 7F11 Monoclonal Antibodies

Chimeric and humanized 7F11 antibodies (7F11-SFD1/K2 and 7F11-SFD2/K2)were produced and purified using the methods described in Example 3, andanalyzed for their ability to bind to CD52 expressed on the surface ofCHO-CD52 cells (CHO cells engineered to express human CD52) by flowcytometry. Briefly, 2×10⁵ CHO-CD52 cells were incubated with an antibodyat 10 μg/ml in PBS containing 5% fetal bovine serum and 5% goat serum.Bound antibody was detected with a FITC-labeled goat anti-humansecondary antibody which detected chimeric or humanized anti-CD52antibodies. Labeled cells were analyzed using a FACSCalibur system(Becton Dickinson) and the data was analyzed using FlowJo version 7.2software (Tree Star, Inc, Oregon, USA). The histogram in FIG. 25compares the levels of CD52 detected with chimeric and humanized 7F11antibodies. The results indicate that the humanized 7F11 antibodiesbound as well or slightly better than the chimeric 7F11 antibody to CD52expressing cells.

Example 23 Humanization of Anti-CD52 Antibody Clone 2C3

Humanization of anti-human CD52 antibody clone 2C3 was performed bygrafting the CDR regions from the mouse 2C3 antibody into a humanantibody variable region framework as described in Example 14 for clone4B10 antibody humanization. CDR-1, CDR-2, and CDR-3 sequences of theheavy chain and light chain of 2C3 were grafted into VH3-72 and VK2 A18bhuman framework regions, respectively. The human JH6 (WGQGTTVTVSS: SEQID NO: 133) and JK5 (FGQGTRLEIK: SEQ ID NO: 135) sequences were selectedas the C-terminal peptides for the humanized heavy and light chains,respectively, to generate humanized heavy chain (2C3-SFD1) and lightchain (2C3-VK1) variable region sequences for 2C3 (FIGS. 26A and B).Unlike humanized clones 4B10 and 7F11, the binding affinity for theCDR-grafted humanized 2C3 antibody was greatly reduced. Binding affinitywas restored by introducing back mutations to the CDR-grafted structure,with the aim of limiting the number of back mutations to a minimum tokeep the reshaped antibody as “human” as possible, thus reducing thepossibility of immunogenicity. Single or multiple back mutations wereincorporated into both the humanized heavy and light chain variableregion sequences. The positions of the back mutations (as denoted by theKabat numbering system) are depicted in Table 10 and Table 11 below.Antibodies generated with these back mutations were evaluated forrestored binding affinity. Three light chain variants (2C3-VK1(L46R),also referred to as 2C3-VK11; 2C3-VK1(Y36L-L46R), also referred to as2C3-VK12; and 2C3-VK1(M4I-A19V-Y36L-Q45K-L46R), also referred to as2C3-VK13) and 5 heavy chain variants (2C3-SFD1(L78V), also referred toas 2C3-VH12; 2C3-SFD1(G49A), also referred to as 2C3-VH15;2C3-SFD1(G49A-L78V), also referred to as 2C3-VH16;2C3-SFD1(L18M-G49A-L78V), also referred to as 2C3-VH17; and2C3-SFD1(L18M-G42E-G49A-L78V), also referred to as 2C3-VH19) weregenerated using standard molecular biology techniques. The amino acidsequences for CDR-grafted heavy chain variable region sequence 2C3-SFD1and back mutants 2C3-VH12, 2C3-VH15, 2C3-VH16, 2C3-VH17, and 2C3-VH19are shown in FIG. 26A with the back mutated amino acids underlined andthe CDRs boldfaced. Similarly, for the light chain sequences,CDR-grafted variable region sequence 2C3-VK1 and back mutants 2C3-VK11,2C3-VK12, and 2C3-VK13 are shown in FIG. 26B with the back mutated aminoacids underlined and the CDRs boldfaced.

The full-length heavy chain amino acid sequence of 2C3-SFD1 (SEQ ID NO:272) and the full-length light chain amino acid sequence of 2C3-K12 (SEQID NO: 273) are shown in FIG. 106.

TABLE 10 2C3 clone heavy chain back mutants Clone ID Mutation (Kabatnumbering position) 2C3-VH12 L to V (78) 2C3-VH15 G to A (49) 2C3-VH16 Gto A (49), L to V (78) 2C3-VH17 L to M (18), G to A (49), L to V (78)2C3-VH19 L to M (18), G to E (42), G to A (49), L to V (78)

TABLE 11 2C3 clone light (kappa) chain back mutants Clone ID Mutation(Kabat numbering position) 2C3-VK11 L to R (46) 2C3-VK12 Y to L (36) andL to R (46) 2C3-VK13 M to I (4), A to V (19), Y to L (36), QL to KR (45,46)

Example 24 Assessment of the Binding Activities of Chimeric andHumanized 2C3 Monoclonal Antibodies

Chimeric and humanized 2C3 antibodies were produced and purified usingthe methods described in Example 3. A number of the humanized antibodiesproduced by pairing heavy chain variants with light chain variants, anda corresponding chimeric antibody, were analyzed by flow cytometry fortheir ability to bind to CD52 expressed on the surface of CHO-CD52cells, using the methods described in Example 22. The binding datasuggest that clones generated by pairing heavy chain variants with lightchain variants 2C3-VK1 or 2C3-VK11 had reduced binding ability, whileclones generated by pairing heavy chain variants with 2C3-VK12 or2C3-VK13 showed binding equivalent to or better than that of a chimeric2C3 antibody. A representative histogram of selected clones (FIG. 27A)compares the level of CD52 detected by chimeric and humanized 2C3antibodies. Binding of 2C3-SFD1/K1 is reduced significantly compared tothat of the corresponding chimeric antibody. Incorporating a singlemouse residue at position 46 (leucine to arginine) in the light chain(resulting in 2C3-VK11) did not restore the binding when paired withheavy chain 2C3-SFD1 to make antibody 2C3-SFD1/K11. Further, binding wasnot restored by incorporating three back mutations in the heavy chain(resulting in 2C3-VH17) to make antibody 2C3-H17/K11. However, bindingwas completely restored when the 2C3-SFD1 heavy chain was paired with2C3-VK12, which has two back mutations, to make antibody 2C3-SFD1/K12,suggesting that specific back mutations need to be incorporated torestore binding avidity. FIG. 27B shows a histogram of selectedhumanized clones that demonstrate binding equivalent to that of achimeric 2C3 antibody. These results indicate that the back mutation oftwo amino acid residues in the 2C3-VK12 light chain was sufficient tocompletely restore antibody avidity. The changes at residues 36 (Y to L)and 46 (L to R) were able to restore binding when paired with almost anyheavy chain variant. As such, the humanized 2C3 clone showing restoredbinding with minimal framework residues derived from the original mouseantibody is 2C3-SFD1/K12.

Example 25 Humanization of Anti-CD52 Antibody Clone 12G6

Humanization of anti-human CD52 antibody clone 12G6 was performed bygrafting the CDR regions from the mouse 12G6 antibody into a humanantibody variable region framework as described in Example 14 for clone4B10 antibody humanization. CDR-1, CDR-2, and CDR-3 sequences of theheavy chain and light chain of 12G6 were grafted into VH3-72 and VK2A18b human framework regions, respectively. The human JH6 (WGQGTTVTVSS:SEQ ID NO: 133) and JK2 (FGQGTKLEIK: SEQ ID NO: 134) sequences wereselected as the C-terminal peptides for the humanized heavy and lightchains, respectively, to generate humanized heavy chain (12G6-SFD1) andlight chain (12G6-VK1) variable region sequences for 12G6 (FIGS. 28A and28B). When the 12G6-SFD1 heavy chain variable region and 12G6-VK1 lightchain variable region were combined in the humanized 12G6-SFD1/K1antibody, the binding affinity for CD52 was greatly reduced. Bindingaffinity was restored by introducing back mutations to the CDR graftedstructure. Single or multiple back mutations were incorporated into boththe humanized heavy and light chain variable region sequences. Thepositions of these back mutations (as denoted by the Kabat numberingsystem) are depicted in Table 12 and Table 13 below. Antibodiesgenerated with these back mutations were evaluated for restored bindingaffinity. Four light chain variants (12G6-VK1(Y36V), also referred to as12G6-VK10; 12G6-VK1(Y36V-Q45K-L46R), also referred to as 12G6-VK11;12G6-VK1(Y36V-L46R), also referred to as 12G6-VK12; and 12G6-VK1(L46R),also referred to as 12G6-VK13) and three heavy chain variants(12G6-SFD1(L78V), also referred to as 12G6-VH10; 12G6-SFD1(G49A), alsoreferred to as 12G6-VH11; and 12G6-SFD1(G49A-L78V), also referred to as12G6-VH12) were generated using standard molecular biology techniques.The amino acid sequences for the CDR grafted heavy chain variable regionsequence 12G6-SFD1 and back mutants 12G6-VH10, 12G6-VH11, and 12G6-VH12are shown in FIG. 28A with the back mutated amino acids underlined andthe CDRs boldfaced. Similarly, for the light chain sequences, CDRgrafted variable region sequence 12G6-VK1 and back mutants 12G6-VK10,12G6-VK11, 12G6-VK12, and 12G6-VK13 are shown in FIG. 28B with the backmutated amino acids underlined and the CDRs boldfaced.

The full-length heavy chain amino acid sequence of 12G6-SFD1 (SEQ ID NO:279) and the full-length light chain amino acid sequence of 12G6-K12(SEQ ID NO: 280) are shown in FIG. 109.

TABLE 12 12G6 clone heavy chain back mutants Clone ID Mutation (Kabatnumbering position) 12G6-VH10 L to V (78) 12G6-VH11 G to A (49)12G6-VH12 G to A (49) and L to V (78)

TABLE 13 12G6 clone light (kappa) chain back mutants Clone ID Mutation(Kabat numbering position) 12G6-VK10 Y to V (36) 12G6-VK11 Y to V (36),QL to KR (45, 46) 12G6-VK12 Y to V (36), L to R (46) 12G6-VK13 L to R(46)

Example 26 Assessment of the Binding Activities of Chimeric andHumanized 12G6 Monoclonal Antibodies

Chimeric and humanized 12G6 antibodies were produced and purified usingthe methods described in Example 3. A number of the humanized antibodiesproduced by pairing heavy chain variants with light chain variants, anda corresponding chimeric antibody, were analyzed by flow cytometry fortheir ability to bind to CD52 expressed on the surface of CHO-CD52cells, using the methods described in Example 22. The binding datasuggest that clones generated by pairing heavy chain variants with lightchain variants 12G6-VK1, 12G6-VK10, or 12G6-VK13 had reduced bindingability, while clones generated by pairing heavy chain variants with12G6-VK11 or 12G6-VK12 showed binding equivalent to or better than thatof the corresponding chimeric 12G6 antibody. A representative histogramof selected clones (FIG. 29) compares the level of CD52 detected bychimeric and humanized 12G6 antibodies. These results indicate that theback mutation of two amino acid residues in the 12G6 light chainvariable region (clone 12G6-VK12) was sufficient to completely restoreantibody specificity. The changes at Kabat numbering residues 36 (Y toV) and 46 (L to R) were able to restore binding when paired with almostany heavy chain variant. As such, the humanized 12G6 clone showingrestored binding with minimal framework residues derived from theoriginal mouse antibody is 12G6-SFD1/K12.

Example 27 Humanization of Anti-CD52 Antibody Clone 9D9

Humanization of anti-human CD52 antibody clone 9D9 was performed bygrafting the CDR regions from the mouse 9D9 antibody into a humanantibody variable region framework as described in Example 14 for clone4B10 antibody humanization. CDR-1, CDR-2, and CDR-3 sequences of theheavy chain and light chain of 9D9 were grafted into VH3-23 and VK2 A18bhuman framework regions, respectively. The human JH6 (WGQGTTVTVSS: SEQID NO: 133) and JK2 (FGQGTKLEIK: SEQ ID NO: 134) sequences were selectedas the C-terminal peptides for the humanized heavy and light chains,respectively, to generate humanized heavy chain (9D9-VH10) and lightchain (9D9-VK2) variable region sequences (FIGS. 30A and 30B). When the9D9-VH10 heavy chain and 9D9-VK2 light chain were combined in thehumanized 9D9-H10/K2 antibody, the binding affinity for CD52 was greatlyreduced. Binding affinity was restored by introducing back mutations tothe CDR grafted structure. Single or multiple back mutations wereincorporated into both the humanized heavy and light chain variableregion sequences. The positions of the back mutations (as denoted by theKabat numbering system) are depicted in Table 14 and Table 15 below.Antibodies generated with these back mutations were evaluated forrestored binding affinity. Four light chain variants(9D9-VK2(Y36L-Q45K-L46R), also referred to as 9D9-VK12;9D9-VK2(Y36L-L46R), also referred to as 9D9-VK13; 9D9-VK2(L46R), alsoreferred to as 9D9-VK14; and 9D9-VK2(Q45K-L46R), also referred to as9D9-VK15) and five heavy chain variants(9D9-VH10(W47L-V48T-549A-N765-L78V), also referred to as 9D9-VH11;9D9-VH10(W47L-V48T-S49A), also referred to as 9D9-VH15; 9D9-VH10(W47L),also referred to as 9D9-VH16; 9D9-VH10(W47L-V48T), also referred to as9D9-VH17; and 9D9-VH10(W47L-S49A), also referred to as 9D9-VH18) weregenerated using standard molecular biology techniques. The amino acidsequences for CDR-grafted heavy chain variable region sequence 9D9-VH10and back mutants 9D9-VH11, 9D9-VH15, 9D9-VH16, 9D9-VH17, and 9D9-VH18are shown in FIG. 30A with the back mutated amino acids underlined andthe CDRs boldfaced. Similarly, for the light chain sequences,CDR-grafted variable region sequence 9D9-VK2 and back mutants 9D9-VK12,9D9-VK13, 9D9-VK14, and 9D9-VK15 are shown in FIG. 30B with the backmutated amino acids underlined and the CDRs boldfaced.

The full-length heavy chain amino acid sequences of 9D9-H16 (SEQ ID NO:276) and 9D9-H18 (SEQ ID NO: 277), and the full-length light chain aminoacid sequence of 9D9-K13 (SEQ ID NO: 278) are shown in FIG. 108.

TABLE 14 9D9 heavy chain back mutants Clone ID Mutation (Kabat numberingposition) 9D9-VH11 WVS to LTA (47-49), N to S (76), L to V (78) 9D9-VH15WVS to LTA (47-49) 9D9-VH16 W to L (47) 9D9-VH17 WV to LT (47, 48)9D9-VH18 W to L (47) and S to A (49)

TABLE 15 9D9 light (kappa) chain back mutants Clone ID Mutation (Kabatnumbering position) 9D9-VK12 Y to L (36) and QL to KR (45, 46) 9D9-VK13Y to L (36) and L to R (46) 9D9-VK14 L to R (46) 9D9-VK15 QL to KR (45,46)

Example 28 Assessment of the Binding Activities of Chimeric andHumanized 9D9 Monoclonal Antibodies

Chimeric and humanized 9D9 antibodies were produced and purified usingthe methods described in Example 3. A number of the humanized antibodiesproduced by pairing heavy chain variants with light chain variants, anda corresponding chimeric antibody, were analyzed by flow cytometry fortheir ability to bind to CD52 expressed on the surface of CHO-CD52 cells(CHO cells engineered to express human CD52), using the methodsdescribed in Example 22. The binding data suggest that clones generatedby pairing heavy chain variants with light chain variants 9D9-VK2,9D9-VK14, or 9D9-VK15 had reduced binding ability, while clonesgenerated by pairing 9D9-VK12 or 9D9-VK13 light chain variants with backmutated heavy chain variants 9D9-VH11, 9D9-VH15, 9D9-VH16, and 9D9-VH18showed binding equivalent to or better than that of the correspondingchimeric 9D9 antibody. When light chain variants 9D9-VK12 and 9D9-VK13were paired with the parental CDR grafted heavy chain 9D9-VH10 or theback mutated 9D9-VH17 sequence, binding was significantly reduced,suggesting that for humanized 9D9 clones, both heavy chain and lightchain sequences have to be engineered with back mutations to restorebinding ability. A representative histogram of selected clones (FIG. 31)compares the level of CD52 detected by chimeric and humanized 9D9antibodies. These results indicate that the back mutation of two aminoacid residues (e.g., Y to L at position 36, and L to R at position 46)in the 9D9 light chain variable region (clone 9D9-VK13) was necessary torestore antibody specificity when paired with heavy chains that weremutated at one position (e.g., W to L, at position 47) or at twopositions (e.g., W to L at position 47 and S to A at position 49). Assuch, the humanized 9D9 clones showing restored binding with minimalframework residues derived from the original mouse antibody are9D9-H16/K13 and 9D9-H18/K13.

Example 29 Determination of Relative Binding Efficiency of HumanizedAnti-Human CD52 Antibodies

The EC₅₀ values of chimeric and humanized anti-CD52 antibodies wereestimated using CD4+ T cells isolated from healthy donor PBMCs obtainedfrom commercial sources (Bioreclamation, NY, USA). CD4+ T cells wereisolated by negative selection using an EasySep kit (Stem CellTechnologies). CD4+ T cells isolated from huCD52 transgenic CD1 mousespleen tissue were also used (Stem Cell Technologies) according to themethods described above in Example 20 for CHO-CD52 cells. Briefly, humanCD4+ T cells were isolated from 50 ml of peripheral blood from healthyvolunteers (Bioreclamation), and huCD52 transgenic mouse CD4+ T cellswere isolated from spleen tissue. Cells were rinsed with PBS/5% FBS anddeposited into round-bottom 96 well plates at 1×10⁵ cells per well.Primary antibody staining was done with an 8 point serial dilution (1:3)of each anti-CD52 chimeric and humanized antibody starting at 100 μg/mL.A FITC-conjugated goat F(ab)₂ fragment of anti-human Fc gamma at 10μg/mL (Jackson 109-096-098) secondary antibody was used. Cells werewashed 3 times in ice-cold PBS/5% FBS before and after each incubation.Cells were fixed with PBS containing 2% methanol-free paraformaldehydeand evaluated by flow cytometry. The flow cytometry data was analyzedusing GraphPad Prism software to determine an EC₅₀ value with 95%confidence interval. Based on the binding of anti-CD52 antibodies toCD4+ T cells isolated from human PBMCs and to CD4+ T cells isolated fromspleen tissue of human CD52 transgenic mice, binding curves (FIGS. 32A,32B, 32C) were generated and EC₅₀ values estimated and shown in FIG. 33.All of the antibodies showed similar binding characteristics to bothhuman CD4+ T cells and to CD4+ T cells isolated from human CD52transgenic mice. Binding data indicate that the humanized antibodieshave equivalent or better binding affinities compared to their parentalchimeric antibodies, suggesting that binding affinity is retained orimproved upon humanization. Humanized 2C3 and 12G6 antibodies have atleast two fold lower EC₅₀ values than a Campath-1H® antibody asdetermined by this cell binding assay.

Example 30 Evaluation of the Binding of Humanized Anti-CD52 Antibodiesto a Defined Lymphocyte Population

Campath-1H® (C1H) and humanized 2C3 (2C3-SFD1/K12), 9D9 (9D9-H16/K13 and9D9-H18/K13), and 12G6 (12G6-SFD1/K11, 12G6-SFD1/K12) antibodies wereevaluated for their binding to various PBMC subsets in normal donorPBMCs using the methods described above in Example 7 for chimericanti-CD52 antibodies. A number of fluorochrome conjugated antibodieswere used for flow cytometric analysis. Anti-CD27-PE, anti-CD19 andanti-CD11c-PE Cy5, anti-CD56 and anti-CD123-PE Cy7, anti-CD16-APC Cy7,and CD4-APC were obtained from BD Biosciences (San Diego, Calif.), whileanti-CD54RA-ECD and anti-HLA-DR-ECD were obtained from Beckman Coulter.Anti-CD3-Pacific Blue, anti-CD8 and anti-CD14-Pacific Orange, andanti-CD4-APC cy5.5 were obtained from Invitrogen (CA). All of thehumanized anti-human CD52 antibodies (9D9-H18/K13, 9D9-H16/K13,12G6-SFD1/K11, 12G6-SFD1/K12, and 2C3-SFD1/K12) as well as theCampath-1H® were conjugated to FITC. Healthy human peripheral bloodmononuclear cells were obtained either from cryopreserved buffy coats orfrom mononuclear cells separated from the blood of normal donorsobtained from commercial vendors (Bioreclamation, NY, USA) as describedabove in Example 7. For enrichment of mononuclear cells, humanperipheral blood was diluted 1:1 with sterile phosphate buffered saline(PBS) and carefully layered over Ficoll-Hypaque (GE HealthcareBio-Sciences, Uppsala, Sweden) and centrifuged for 30 min at roomtemperature. The interphase layer of mononuclear cells was drawn out andwashed in PBS containing 5% fetal bovine serum (FACS buffer).Contaminating red blood cells (RBCs) were lysed with RBC lysing solution(Sigma, St. Louis, Mo., USA). Cells were resuspended in cold FACS bufferand the debris was removed using a 40 μm filter. Multi color flowcytometry was performed to evaluate the binding ability of humanizedanti-human CD52 antibodies 2C3 (2C3-SFD1/K12), 9D9 (9D9-H16/K13 and9D9-H18/K13) and 12G6 (12G6-SFD1/K11 and 12G6-SFD1/K12) as compared toCampath-1H®.

Briefly, replicates of 1×10⁶ PBMCs in FACS buffer were incubated withcocktails of pre-titrated dilutions of antibodies to examine eitherlymphocyte or myeloid derived cells. The lymphocyte cocktail comprisedantibodies against CD3, CD27, CD45RA, CD56, CD19, CD8, CD4, and CD16.The antibody cocktail to define myeloid populations included antibodiesagainst HLA-DR, CD11c, CD123, CD4, and CD14. In each of the cocktails,one of the anti-CD52 antibodies was included at 10 μg/ml concentration.The cells were stained for 30 min at 4° C. and were washed and fixed inPBS containing 1% paraformaldehyde. 100,000 events of the stained cellswere acquired on a BD LSR II flow cytometer (BD Biosciences, San Jose,Calif.), and the data was analyzed using FlowJo version 7.2 software(Tree Star, Inc, Oregon, USA). Multiple subsets with distinct phenotypiccharacteristics have been defined among B and T lymphocytes, and CD52has been shown to be expressed on all human lymphocytes. Multi colorflow cytometry analysis was performed to identify the lymphocytesubsets, and to assess similarities and differences in the bindingcharacteristics of the humanized anti-CD52 antibodies to cell surfaceCD52 on defined subsets. Using a combination of markers, phenotypicallydistinct cell populations corresponding to B, T, NK and antigenpresenting cell lineages were first defined. The intensity of staining,which corresponds to the ability of humanized anti-CD52 antibodies todetect CD52 expression on each of the defined cell populations, wasassessed and compared to that of Campath-1H®. The histograms (FIG. 34)compare the level of CD52 detected by each antibody on individualpopulations. The results indicate that all of the humanized anti-CD52antibodies bind to cell surface CD52 to a similar extent. Further, nodifferences were observed between Campath-1H® and humanized anti-CD52antibodies with respect to the level of detection of cell surface CD52.Analysis was performed on six different donors. Representative datagenerated using cells derived from one donor is shown in FIG. 34. Asimilar binding pattern was observed with cells from other donors.

Example 31 Assessment of the ADCC Activities of Chimeric and Humanized7F11 Monoclonal Antibodies

Humanized and chimeric 7F11 antibodies were evaluated for their abilityto mediate ADCC killing of CD52 expressing cells. An ADCC assay wascarried out using the methods described above in Example 6. Briefly, CHOK1 cells engineered to express CD52 protein (CHO-CD52) were used astarget cells. The target cells were labeled with Na₂ ⁵¹CrO₄ (New EnglandNuclear, Boston, Mass.) at 37° C. for 2-3 hrs. The cells were washed,re-suspended in RPMI 1640 media with 10% FCS, and mixed with an IgGcontrol antibody, a chimeric 7F11 antibody, or a humanized 7F11 antibody(7F11-SFD1/K2 or 7F11-SFD2/K2) at various concentrations ranging from 5μg/ml to 0.01 μg/ml. Human NK cells isolated from PBMCs using an NK cellisolation kit (Stem Cell Technologies) were used as effector cells andwere added at a 1:5 target to effector cell ratio. After 2-6 hrsincubation, 25 μl of cell-free supernatant were collected from each welland counted in a MicroBeta Trilux Scintillation Counter (Wallac,Gaithersburg, Md.). The amount of ⁵¹Cr spontaneously released wasobtained by incubating target cells in medium alone. Spontaneous releasefrom target cells was typically less than 20%. The total amount of ⁵¹Crincorporated was determined by adding 1% Triton X-100 in distilledwater, and the percentage lysis was calculated as follows: [(samplec.p.m.−spontaneous c.p.m.)/(total c.p.m.−spontaneous c.p.m.)]×100. FIG.35 illustrates the concentrations of control IgG, chimeric 7F11antibody, and humanized 7F11 antibodies used in the assay (X axis) vs. %specific lysis (Y axis). The results indicate that humanized 7F11antibodies (7F11-SFD1/K2 and 7F11-SFD2/K2) mediated equivalent orslightly better ADCC killing as compared to a chimeric 7F11 antibody.The control IgG1 isotype showed only low levels of background killing atthe concentrations tested.

Example 32 Assessment of the CDC Activities of Chimeric and Humanized7F11 Monoclonal Antibodies

Humanized and chimeric 7F11 antibodies were evaluated for their abilityto mediate complement dependent cytotoxicity (CDC) of CD52 expressingcells. A CDC assay was carried out using the methods described above inExample 5 for chimeric anti-CD52 antibodies. Briefly, CHO K1 cellsengineered to express CD52 protein (CHO-CD52) were used as target cellsand labeled with Na₂ ⁵¹CrO₄ (New England Nuclear, Boston, Mass.) at 37°C. for 2-3 hrs. The cells were washed, resuspended in RPMI 1640 media,and mixed with an IgG control antibody, a chimeric 7F11 antibody, or ahumanized 7F11 antibody (7F11-SFD1/K2 or 7F11-SFD2/K2) at variousconcentrations ranging from 20 μg/ml to 500 ng/ml. Human complement(Sigma) was added to the experimental wells to a final concentration of10%. After a 1-5-hour incubation, 25 μl of cell-free supernatant werecollected from each well and counted in a MicroBeta Trilux ScintillationCounter (Wallac, Gaithersburg, Md.). The amount of ⁵¹Cr spontaneouslyreleased was obtained by incubating target cells in medium alone.Spontaneous release from target cells was typically less than 20%. Thetotal amount of ⁵¹Cr incorporated was determined by adding 1% TritonX-100 in distilled water, and the percentage lysis was calculated asfollows: [(sample counts per minute (c.p.m.)−spontaneous c.p.m.)/(totalc.p.m.−spontaneous c.p.m.)]×100. FIG. 36 illustrates the concentrationsof control IgG, chimeric 7F11 antibody, and humanized 7F11 antibodies(7F11-SFD1/K2 and 7F11-SFD2/K2) used in the assay (X axis) vs. %specific lysis (Y axis). The results indicate that the chimeric 7F11antibody and humanized antibody 7F11-SFD1/K2 mediated equivalentkilling, while humanized antibody 7F11-SFD2/K2 mediated significantlybetter CDC killing than the chimeric 7F11 antibody. The control IgG1isotype antibody showed only low levels of background killing at theconcentrations tested.

Example 33 Assessment of the ADCC Activities of Chimeric and Humanized2C3 Monoclonal Antibodies

Humanized and chimeric 2C3 antibodies were evaluated for their abilityto mediate ADCC killing of CD52 expressing cells. An ADCC assay wascarried out using the methods described above in Example 6, with slightmodifications. Briefly, T cells isolated from healthy donor PBMCs usinga CD4+ T cell isolation kit (Stem Cell Technologies) were used as targetcells. The target cells were labeled overnight with Na₂ ⁵¹CrO₄ (NewEngland Nuclear, Boston, Mass.) at 37° C. The cells were washed,re-suspended in RPMI 1640 media with 10% FCS, and mixed with an IgGcontrol antibody, a chimeric 2C3 antibody, or a humanized 2C3 antibody(2C3-SFD1/K12) at various concentrations ranging from 10 μg/ml to 100pg/ml. Human NK cells isolated from PBMCs (using an NK cell isolationkit from Stem Cell Technologies) were used as effector cells and wereadded at a 1:5 target to effector cell ratio. After 2-6 hrs ofincubation, 25 μl of cell-free supernatant were collected from each welland counted in a MicroBeta Trilux Scintillation Counter (Wallac,Gaithersburg, Md.). The amount of ⁵¹Cr spontaneously released wasobtained by incubating target cells in medium alone. Spontaneous releasefrom target cells was typically less than 20%. The total amount of ⁵¹Crincorporated was determined by adding 1% Triton X-100 in distilledwater, and the percentage lysis was calculated as follows: [(samplec.p.m.−spontaneous c.p.m.)/(total c.p.m.−spontaneous c.p.m.)]×100. FIG.37 illustrates the concentrations of control IgG, chimeric 2C3 antibody,and humanized 2C3 antibody (2C3-SFD1/K12) used in the assay (X axis) vs.% specific lysis (Y axis). The results indicate that the humanized 2C3antibody 2C3-SFD1/K12 mediated ADCC killing equivalent to that of the2C3 chimeric antibody. The IgG1 isotype control showed only low levelsof background killing at the concentrations tested.

Example 34 Assessment of the CDC Activities of Chimeric and Humanized2C3 Monoclonal Antibodies

Humanized and chimeric 2C3 antibodies were evaluated for their abilityto mediate complement dependent cytotoxicity (CDC) of CD52 expressingcells. A CDC assay was carried out using the methods described above inExample 5, with slight modifications. Briefly, T cells isolated fromhealthy donor PBMCs were used as target cells and labeled overnight withNa₂ ⁵¹CrO₄ (New England Nuclear, Boston, Mass.) at 37° C. Afterovernight labeling, the cells were washed, re-suspended in RPMI 1640media with 10% FCS, and mixed with an IgG control antibody, a chimeric2C3 antibody, or a humanized 2C3 antibody (2C3-SFD1/K12) at variousconcentrations ranging from 10 μg/ml to 10 ng/ml. Human complement(Sigma) was added to the experimental wells to a final concentration of10%. After a 1-5 hour incubation, 25 μl of cell-free supernatant werecollected from each well and counted in a MicroBeta Trilux ScintillationCounter (Wallac, Gaithersburg, Md.). The amount of ⁵¹Cr spontaneouslyreleased was obtained by incubating target cells in medium alone.Spontaneous release from target cells was typically less than 20%. Thetotal amount of ⁵¹Cr incorporated was determined by adding 1% TritonX-100 in distilled water, and the percentage lysis was calculated asfollows: [(sample counts per minute (c.p.m.)−spontaneous c.p.m.)/(totalc.p.m.−spontaneous c.p.m.)]×100. FIG. 38 illustrates the concentrationsof control IgG, chimeric 2C3 antibody, and humanized 2C3 antibody(2C3-SFD1/K12) used in the assay (X axis) vs. % specific lysis (Y axis).The results indicate that the chimeric 2C3 antibody and the humanized2C3 antibody (2C3-SFD1/K12) mediated equivalent lysis. The control IgG1isotype antibody showed only low levels of background killing at theconcentrations tested.

Example 35 Assessment of the ADCC Activities of Chimeric and Humanized12G6 Monoclonal Antibodies

Humanized and chimeric 12G6 antibodies were evaluated for their abilityto mediate ADCC killing of CD52 expressing cells. An ADCC assay wascarried out by chromium release assays using T cells isolated fromhealthy donor PBMCs as target cells, as described above in Example 31.FIG. 39 illustrates the concentrations of control IgG, chimeric 12G6antibody, and humanized 12G6 antibodies (12G6-SFD1/K11 or 12G6-SFD1/K12)used in the assay (X axis) vs. % specific lysis (Y axis). The resultsindicate that humanized 12G6 antibodies 12G6-SFD1/K11 and 12G6-SFD1/K12mediated equivalent ADCC killing as compared to the 12G6 chimericantibody. The IgG1 isotype control showed only low levels of backgroundkilling at the concentrations tested.

Example 36 Assessment of the CDC Activities of Chimeric and Humanized12G6 Monoclonal Antibodies

Humanized and chimeric 12G6 antibodies were evaluated for their abilityto mediate complement dependent cytotoxicity (CDC) of CD52 expressingcells. A CDC assay was carried out by chromium release assays using Tcells isolated from healthy donor PBMCs as target cells, as describedabove in Example 32. FIG. 40 illustrates the concentrations of controlIgG, chimeric 12G6 antibody, and humanized 12G6 antibodies(12G6-SFD1/K11 and 12G6-SFD1/K12) used in the assay (X axis) vs. %specific lysis (Y axis). The results indicate that the chimeric 12G6antibody mediated equivalent lysis as compared to humanized 12G6antibodies (12G6-SFD1/K11 and 12G6-SFD1/K12). The control IgG1 isotypeantibody showed only low levels of background killing at theconcentrations tested.

Example 37 Assessment of the ADCC Activities of Chimeric and Humanized9D9 Monoclonal Antibodies

Humanized and chimeric 9D9 antibodies were evaluated for their abilityto mediate ADCC killing of CD52 expressing cells. An ADCC assay wascarried out by chromium release assays using T cells isolated fromhealthy donor PBMCs as target cells, as described above in Example 31.FIG. 41 illustrates the concentrations of control IgG, chimeric 9D9antibody, and humanized 9D9 antibodies (9D9-H10/K13, 9D9-H11/K13,9D9-H16/K13, and 9D9-H18/K13) used in the assay (X axis) vs. % specificlysis (Y axis). The results indicate that the chimeric and humanized 9D9antibodies (with the exception of 9D9-H10/K13) mediated equivalent ADCCkilling. The IgG1 isotype control showed only low levels of backgroundkilling at the concentrations tested.

Example 38 Assessment of the CDC Activities of Chimeric and Humanized9D9 Monoclonal Antibodies

Humanized and chimeric 9D9 antibodies were evaluated for their abilityto mediate complement dependent cytotoxicity (CDC) of CD52 expressingcells. A CDC assay was carried out by chromium release assays using Tcells isolated from healthy donor PBMCs as target cells, as describedabove in Example 32. FIG. 42 illustrates the concentrations of controlIgG, chimeric 9D9 antibody, and humanized 9D9 antibodies (9D9-H10/K13,9D9-H11/K13, 9D9-H16/K13, and 9D9-H18/K13) used in the assay (X axis)vs. % specific lysis (Y axis). The results indicate that a chimeric 9D9antibody mediated equivalent lysis as compared to humanized 9D9antibodies (with the exception of 9D9-H10/K13). The control IgG1 isotypeantibody showed only low levels of background killing at theconcentrations tested.

Example 39 Assessment of the ADCC Activities of Campath-1H® andHumanized Anti-CD52 Antibodies on Primary T Cells

Campath-1H® and humanized anti-CD52 antibodies were evaluated for theirability to mediate ADCC killing of CD52 expressing cells. An ADCC assaywas carried out by chromium release assays using T cells isolated fromhealthy donor PBMCs as target cells, as described above in Example 31.FIG. 43 illustrates the concentrations of control IgG, Campath-1H®, andhumanized 2C3-SFD1/K12, 9D9-H16/K13, 9D9-H18/K13, 12G6-SFD1/K11, and12G6-SFD1/K12 antibodies used in the assay (X axis) vs. % specific lysis(Y axis). The results indicate that the above humanized 2C3, 9D9, and12G6 antibodies mediated ADCC killing equivalent to that of Campath-1H®at concentrations in excess of 10 ng/ml. The IgG1 isotype control showedonly low levels of background killing at the concentrations tested.

Example 40 Assessment of the CDC Activities of Campath-1H® and HumanizedAnti-CD52 Antibodies on Primary T Cells

Campath-1H® and humanized anti-CD52 antibodies were evaluated for theirability to mediate complement dependent cytotoxicity (CDC) of CD52expressing cells. A CDC assay was carried out by chromium release assaysusing T cells isolated from healthy donor PBMCs as target cells, asdescribed above in Example 32. FIG. 44 illustrates the concentrations ofcontrol IgG, Campath-1H®, and humanized 2C3-SFD1/K12, 9D9-H16/K13,9D9-H18/K13, 12G6-SFD1/K11, and 12G6-SFD1/K12 antibodies used in theassay (X axis) vs. % specific lysis (Y axis). The results indicate thathumanized 2C3 and 12G6 antibodies mediated CDC killing equivalent toCampath-1H®, while humanized 9D9 antibodies demonstrated significantlyreduced CDC activity, similar to their corresponding chimeric antibody.The IgG1 isotype control showed only low levels of background killing atthe concentrations tested.

Example 41 Assessment of Neutralizing Ability of Serum SamplesContaining Anti-Campath-1H® Neutralizing Antibodies to Block Humanized2C3, 12G6, and 9D9 Anti-CD52 Antibody Activity

To assess the ability of humanized antibodies to bind to CD52 expressingcells in the presence of neutralizing antibodies against Campath-1H®,anti-CD52 antibodies (Campath-1H®, 2C3-SFD1/K12, 9D9-H16/K13 and12G6-SFD1/K12) were reacted with human serum containing anti-Campath-1H®antibody reactivity and evaluated for binding to CD52 expressing Rajicells. Serum samples obtained from relapsing remitting multiplesclerosis patients who were enrolled in the CAMMS223 study (The CAMMS223Trial Investigators, “Alemtuzumab vs. interferon Beta-1a in earlymultiple sclerosis,” N Engl J Med 359:1786-1801 (2008)) were used in theassay. Repeated administration of the Campath-1H® antibody resulted ingeneration of anti-Campath-1H® antibody responses in most patients. Theanti-Campath-1H antibody titer is very low at month 12 in most patients,and increased significantly upon administration of a second cycle ofCampath-1H® resulting in a high titer anti-Campath-1H® response in thesera at month 13. Anti-CD52 antibody neutralization assays were carriedout using month 12 and month 13 serum samples obtained from fivedifferent MS patients (MS-1 to MS-5) who had been treated withCampath-1H® under the CAMMS223 protocol. FITC-conjugated anti-CD52antibodies Campath-1H®, 2C3-SFD1/K12, 12G6-SFD1/K12, and 9D9-H16/K13(used in Example 30 and shown to bind to CD52 expressing cells) wereused to stain Raji cells that express human CD52 in the absence orpresence of a range of dilutions of serum obtained from patients whohave been treated with Campath-1H. Briefly, MS patient serum samples(month 12 and month 13) were made into 6 fold serial dilutions andincubated with 10 μg/ml of FITC-conjugated anti-CD52 antibodies(Campath-1H®, 2C3-SFD1/K12, 12G6-SFD1/K12, and 9D9-H16/K13) for 1 hr at37° C. Raji cells were rinsed with a staining buffer containing HBSS, 5%FBS, and 0.1% azide, and then deposited into round-bottom 96 well platesat 1×10⁵ cells per well. Cells were blocked with 10 μg/ml of human IgGFc fragment for 30 min on ice in staining buffer. The cells were thenwashed with staining buffer and re-suspended in 100 μl of theantibody-serum mix as described above. After 30 minutes on ice, cellswere washed and fixed with BD Cytofix and the FITC-labeled antibodycoated cells were analyzed using a FACSCalibur system (BectonDickinson), after which the data was analyzed using FlowJo version 7.2software (Tree Star, Inc, Oregon, USA). Binding of FITC-conjugatedanti-CD52 antibodies in the presence of anti-Campath-1H® neutralizingantibodies in the serum was assessed by flow cytometry and % bindingrelative to control, as a measure of inhibition was calculated as (MFIwith serum/MFI control (no serum))×100. Representative data from one ofthe donors (MS-1) is shown in FIG. 45. The X axis denotes the serumdilution factor and the Y axis denotes the % control binding as ameasure of antibody neutralizing activity. The data clearly demonstratethat month 12 serum samples have no inhibitory effect on Campath-1H® orother anti-CD52 antibodies, suggesting that there are low or noanti-Campath-1H® blocking antibodies in the serum. Month 13 serumsamples mediated complete inhibition of Campath-1H® binding even at a1:1000 dilution of serum, but did not mediate inhibition of 2C3, 12G6,and 9D9 humanized anti-CD52 antibodies even at the highest concentration(1:24 dilution) tested. Two of the five patients developedanti-Campath-1H® neutralizing antibody titers of >1:1000, whereas threeother patients had about 1:100 Campath-1H® neutralizing antibody titers.Even though two of the patients' month 13 sera had relatively highneutralizing antibody titers of >1:1000 against Campath-1H®, these seradid not inhibit binding of humanized 2C3-SFD1/K12, 12G6-SFD1/K12, and9D9-H16/K13 antibodies, suggesting that the anti-Campath-1H® antibodyreactivity in patients treated with Campath-1H® did not block binding ofthese humanized antibodies to CD52 as presented on cells.

Example 42 Analysis of Depletion and Repopulation of Anti-CD52Antibodies in huCD52 Transgenic Mice (4B10-H1/K1)

The depleting activities of Campath-1H® and the humanized anti-CD52antibody (4B10-H1/K1) at different dose levels were examined in thehuCD52 transgenic mouse. Mice were injected intravenously with 0.1, 0.5,1.0 or 5.0 mg/kg of each antibody. Two hours post dosing, serum wascollected to examine the level of circulating cytokines. Three days postdosing, mice were sacrificed, and blood and spleens were collected fromeach mouse (N=5) to determine the level of cell depletion using flowcytometry analysis. Samples were evaluated to determine the relativenumbers of total T helper cell (CD4+), cytotoxic T cell (CD8+), B cell(B220+) and myeloid cell subpopulations present in the circulatingperipheral blood or spleen of huCD52 transgenic mice. In addition, T andB cell subset analysis was performed to determine the overall depletingeffect. A subset of mice (N=5) were kept alive to monitor therepopulation kinetics. Depletion was greatest in the T cell compartmentwith CD4+ T cells being depleted most followed by CD8+ T cells, B cells,NK cells, and other myeloid cells. Within the CD4+ T cell compartment,naïve CD4+ T cells were depleted the most followed by CD4+ centralmemory (CM), CD4+ effector memory (EM), and CD4+ regulatory T cells(Treg). A similar pattern was observed for CD8+ T cells (Naïve>CM>EM).Conversely, mature B cells were depleted to a greater extent thanimmature B cells. Comparison of Campath-1H® treated mice to 4B10-H1/K1treated mice demonstrated similar patterns of cells in both the bloodand spleen at each of the doses examined.

Serum cytokine analysis demonstrated dose dependent increases for TNFα,IL-6 and MCP-1. The circulating level of these cytokines remainedelevated compared to untreated mice at the 0.5 and 0.1 mg/kg doses aswell. Slight increases were also observed for IL-10 in the Campath-1H®treated group at the three highest doses but only for the highest doseof the humanized 4B10-H1/K1 treated group. No significant increases inthe level of circulating IL-12 or IFNg (not shown) were noted.

By 50-60 days post dosing, with the exception of the 1.0 mg/kg group,lymphocyte levels in all of the Campath-1H® dosed groups had reboundedto the levels of untreated mice. In the 1.0 mg/kg group, lymphocytes hadreturned to normal levels by 80 days post dosing. Similar repopulationkinetics were also observed for the humanized 4B10-H1/K1 antibodytreated mice. Lymphocytes had rebounded to control levels by 50 dayspost dosing in all 4B10-H1/K1 treated groups with the exception of the0.5 mg/kg level. Levels of circulating lymphocytes in the 0.5 mg/kggroup remained decreased throughout the course of the monitoring period.Total lymphocytes were monitored for repopulation in the blood.

FIGS. 46A-46E show the level of CD4+ T cells, CD8+ T cells and B220+ Bcells in the blood 72 hours after dosing with Campath-1H® (“Campath”)and humanized 4B10-H1/K1 (“4B10”) antibodies. FIGS. 47A-47E show thelevel of CD4+ T cells, CD8+ T cells and B220+ B cells in the spleen 72hours after dosing with Campath-1H® (“Campath”) and humanized 4B10-H1/K1(“4B10”) antibodies. FIGS. 48A-48E show the levels of circulatingcytokines 2 hours after dosing with Campath-1H® (“Campath”) andhumanized 4B10-H1/K1 (“4B10”) antibodies. FIGS. 49A-49B show therepopulation of circulating lymphocytes over a timecourse after dosingwith Campath-1H® (“Campath”) and humanized 4B10-H1/K1 (“4B10”)antibodies.

Example 43 Analysis of Depletion and Repopulation of Anti-CD52Antibodies in huCD52 Transgenic Mice (7F11-SFD1/K2 and 7F11-SFD2/K2)

The depleting activity of humanized antibodies (7F11-SFD1/K2 and7F11-SFD2/K2) at different dose levels was examined in huCD52 transgenicmice. Mice were injected intravenously with 0.1, 0.5, 1.0 or 5.0 mg/kgof each antibody. Two hours post dosing, serum was collected to examinethe level of circulating cytokines. Three days post dosing, mice weresacrificed, and blood and spleens were collected from each mouse (N=5)to determine the level of cell depletion using flow cytometry analysis.Samples were evaluated to determine the relative numbers of total Thelper cell (CD4+), cytotoxic T cell (CD8+), B cell (B220+) and myeloidcell subpopulations present in the circulating peripheral blood orspleen of huCD52 transgenic mice In addition, T and B cell subsetanalysis was performed to determine the overall depleting effect. Asubset of mice (N=5) were kept alive to monitor the repopulationkinetics. Administration of each humanized 7F11 antibody (7F11-SFD1/K2and 7F11-SFD2/K2) at all doses resulted in depletion of a significantnumber of both T cells and B cells in the blood. These data alsodemonstrated that various T and B cell subsets are depleted to differingdegrees depending on the dose of antibody used. Naïve T cells (both CD4and CD8) demonstrated the most depletion with other cell populations(including memory and T reg cells) being depleted to a lesser degree. Inthe B cell compartment, mature B cells were depleted more readily thanimmature B cells. In the spleen, dose dependent depletion was observedwith significant depletion of lymphocytes being observed at the 5 and 1mg/kg dose levels. Similar to the case with blood, naïve T cells weremore readily depleted than memory cells. B cells were depleted to alesser extent than T cells with each of the humanized 7F11 clones(7F11-SFD1/K2 and 7F11-SFD2/K2). Depletion was not observed for NK cellsor neutrophils in the blood or the spleen at any of the doses injected.Serum cytokine analysis demonstrated dose dependent increases for bothTNFα and IL-6. Levels of these cytokines remained elevated compared tountreated mice at the 0.5 and 0.1 mg/kg doses as well. Dose dependentincreases in the level of circulating MCP-1 were also noted.

By 30 days post dosing, lymphocyte levels in the 0.5 and 0.1 mg/kg dosedgroups had rebounded to the levels of untreated mice. In the 1.0 and 5.0mg/kg groups, lymphocytes had returned to normal levels by 50 and 80days, respectively, for clone 7F11-SFD1/K2 and by 80 days post dosingfor both the 1.0 and 5.0 mg/kg groups of clone 7F11-SFD2/K2. Totallymphocytes were monitored for repopulation in the blood.

FIGS. 50A-50E show the level of CD4+ T cells, CD8+ T cells and B220+ Bcells in the blood 72 hours after dosing with the humanized 7F11-SFD1/K2(“7F11 SFD1”) and 7F11-SFD2/K2 (“7F11 SFD2”) antibodies. FIGS. 51A-51Eshow the level of CD4+ T cells, CD8+ T cells and B220+ B cells in thespleen 72 hours after dosing with the humanized 7F11-SFD1/K2 (“7F11SFD1”) and 7F11-SFD2/K2 (“7F11 SFD2”) antibodies. FIGS. 52A-52F show thelevels of circulating cytokines 2 hours after dosing with the humanized7F11-SFD1/K2 (“7F11 SFD1”) and 7F11-SFD2/K2 (“7F11 SFD2”) antibodies.FIGS. 53A-53B show the repopulation of circulating lymphocytes over atimecourse after dosing with the humanized 7F11-SFD1/K2 (“7F11 SFD1”)and 7F11-SFD2/K2 (“7F11 SFD2”) antibodies.

Example 44 Analysis of 7F11 Humanized Anti-CD52 Antibodies in CD52Transgenic Mice (7F11-SFD1/K2 and 7F11-SFD2/K2)

The depleting activity of the chimeric 7F11 antibodies and humanized7F11-SFD1/K2 and 7F11-SFD2/K2 antibodies in comparison to Campath-1H®was examined in the huCD52 transgenic mouse. Mice were injectedintravenously with 1.0 mg/kg of each antibody. Three days post dosing,mice were sacrificed, and blood and spleens were collected from eachmouse (N=5) to determine the level of cell depletion using flowcytometry analysis. Samples were evaluated to determine the relativenumbers of total T helper cell (CD4+), cytotoxic T cell (CD8+), B cell(B220+) and myeloid cell subpopulations present in the circulatingperipheral blood or spleen of huCD52 transgenic mice Administration ofCampath-1H® resulted in depletion of a significant number of both Tcells and B cells in the blood and spleen. Although a comparable levelof T cell depletion was observed in the blood for both the chimeric andhumanized 7F11 antibodies (7F11-SFD1/K2 and 7F11-SFD2/K2), B cells weredepleted to a lesser extent. This observation was also apparent in thespleen, where significant T cell depletion was noted, but only a modestlevel of B cell depletion was achieved with the 7F11 antibodies(7F11-SFD1/K2 and 7F11-SFD2/K2).

FIGS. 54A-54B show the level of CD4+ T cells, CD8+ T cells and B220+ Bcells in the blood 72 hours after dosing with Campath-1H® (“Campath”),7F11-chimeric antibodies, and humanized 7F11-SFD1/K2 and 7F11-SFD2/K2antibodies.

Example 45 Analysis of PK Profiles of Anti-CD52 Antibodies in CD52Transgenic Mice (7F11-SFD1/K2 and 7F11-SFD2/K2)

To ensure that the humanization process did not alter the clearance rateof the antibody, the pharmacokinetic profile of the chimeric 7F11anti-CD52 antibody and humanized 7F11-SFD1/K2 and 7F11-SFD2/K2 anti-CD52antibodies was determined in huCD52 transgenic mice. Mice were injectedintravenously with antibodies at 5 mg/kg and blood was collected atvarious timepoints beginning two hours post dosing. The circulatinglevels of each antibody were evaluated using an anti-human IgG ELISA.For each of the humanized clones, there was a slight difference in theCmaxnoted at 2 hours post dosing. Clearance rates for the chimeric 7F11antibody and humanized 7F11-SFD1/K2 and 7F11-SFD2/K2 antibodies weresimilar to each other as well as to Campath-1H® over the course of theexperiment, indicating that the humanization process did notsignificantly alter the pharmacokinetic profile of the antibodies.

FIG. 55 shows the level of Campath-1H® (“Campath”), 7F11-chimericantibody and humanized 7F11-SFD1/K2 and 7F11-SFD2/K2 antibodies in theblood over a timecourse after dosing.

Example 46 Analysis of Depletion and Repopulation of Anti-CD52Antibodies in huCD52 Transgenic Mice (2C3-SFD1/K12)

The depleting activity of the 2C3-SFD1/K12 clone at different doselevels was examined in the huCD52 transgenic mouse. Mice were injectedintravenously with 0.1, 0.5, 1.0 or 5.0 mg/kg of antibody. Two hourspost dosing, serum was collected to potentially examine the level ofcirculating cytokines. Three days post dosing, mice were sacrificed, andblood and spleens were collected from each mouse (N=5) to determine thelevel of cell depletion using flow cytometry analysis. Samples wereevaluated to determine the relative numbers of total T helper cell(CD4+), cytotoxic T cell (CD8+), B cell (B220+) and myeloid cellsubpopulations present in the circulating peripheral blood or spleen ofhuCD52 transgenic mice. In addition, T and B cell subset analysis wasperformed to determine the overall depleting effect. A subset of mice(N=5) were kept alive to monitor the repopulation kinetics.Administration of 2C3-SFD1/K12 at the 5, 1, and 0.5 mg/kg doses resultedin depletion of a significant number of both T cells and B cells in theblood. A variable level of lymphocyte depletion was observed in theblood at the 0.1 mg/kg dose with CD4+ T cells and B cells being depletedto a greater extent than CD8+ T cells. These data also demonstrated thatvarious T and B cell subsets are depleted to differing degrees dependingon the dose of antibody used. Naïve T cells (both CD4 and CD8)demonstrated the most depletion compared to other cell populations(including memory and T reg cells), which were depleted to a lesserdegree. In the B cell compartment, mature B cells were depleted morereadily than immature B cells. In the spleen, dose dependent depletionwas observed with significant depletion of lymphocytes being observed atthe 5 and 1 mg/kg dose levels. Similar to Campath-1H®, naïve T cellswere more readily depleted than memory cells. Depletion was observed forNK cells and neutrophils in the blood, but little to no depletion wasobserved in the spleen at any of the doses injected. Serum cytokineanalysis demonstrated dose dependent increases for both TNFα and IL-6with the 5 mg/kg dose inducing the highest level of each cytokine.Levels comparable to untreated mice were observed in the 0.5 and 0.1mg/kg dose levels for TNFα and the 0.1 mg/kg dose level for IL-6. Dosedependent increases in the level of circulating MCP-1 were also noted.

By 30 days post dosing, lymphocyte levels for the 0.1 and 0.5 mg/kggroups had rebounded to the levels of untreated mice. In the 1.0 and 5.0mg/kg groups, lymphocytes had returned to normal levels by 80 days postdosing. Total lymphocytes were monitored for repopulation in the blood.

FIGS. 56A-56E show the level of CD4+ T cells, CD8+ T cells and B220 Bcells in the blood 72 hours after dosing with 2C3-SFD1/K12 antibodies.FIGS. 57A-57E show the level of CD4+ T cells, CD8+ T cells and B220+ Bcells in the spleen 72 hours after dosing with 2C3-SFD1/K12 antibodies.FIGS. 58A-58F show the levels of circulating cytokines 2 hours afterdosing with 2C3-SFD1/K12 antibodies. FIG. 59 shows the repopulation ofcirculating lymphocytes over a timecourse after dosing with 2C3-SFD1/K12antibodies.

Example 47 Analysis of Depletion and Repopulation of Anti-CD52Antibodies in huCD52 Transgenic Mice (12G6-SFD1/K11)

The depleting activity of the 12G6-SFD1/K11 clone at different doselevels was examined in the huCD52 transgenic mouse. Mice were injectedintravenously with 0.1, 0.5, 1.0 or 5.0 mg/kg of antibody. Two hourspost dosing, serum was collected to potentially examine the level ofcirculating cytokines. Three days post dosing, mice were sacrificed, andblood and spleens were collected from each mouse (N=5) to determine thelevel of cell depletion using flow cytometry analysis. Samples wereevaluated to determine the relative numbers of total T helper cell(CD4+), cytotoxic T cell (CD8+), B cell (B220+) and myeloid cellsubpopulations present in the circulating peripheral blood or spleen ofhuCD52 transgenic mice. In addition, T and B cell subset analysis wasperformed to determine the overall depleting effect. A subset of mice(N=5) were kept alive to monitor the repopulation kinetics.Administration of 12G6-SFD1/K11 at the 5, 1, and 0.5 mg/kg dosesresulted in depletion of a significant number of both T cells and Bcells in the blood. A variable level of lymphocyte depletion wasobserved in the blood at the 0.1 mg/kg dose with CD4+ T cells and Bcells being depleted to a greater extent than CD8+ T cells. These dataalso demonstrated that various T and B cell subsets are depleted todiffering degrees depending on the dose of antibody used. Naïve T cells(both CD4 and CD8) demonstrated the most depletion compared to othercell populations (including memory and T reg cells), which were depletedto a lesser degree. In the B cell compartment, mature B cells weredepleted more readily than immature B cells. In the spleen, dosedependent depletion was observed with significant depletion oflymphocytes being observed at the 5 and 1 mg/kg dose levels. Similar toCampath-1H®, naïve T cells were more readily depleted than memory cells.Depletion was observed for NK cells and neutrophils in the blood butlittle to no depletion was observed in the spleen at any of the dosesinjected. Serum cytokine analysis demonstrated dose dependent increasesfor both TNFα and IL-6 with the 5 mg/kg dose inducing the highest levelof each cytokine. Levels comparable to untreated mice were observed inthe 0.5 and 0.1 mg/kg dose levels for TNFα and the 0.1 mg/kg dose levelfor IL-6. Dose dependent increases in the level of circulating MCP-1were also noted.

By 30 days post dosing, lymphocyte levels had rebounded to the levels ofuntreated mice. In the 1.0 and 5.0 mg/kg groups, lymphocytes hadreturned to normal levels by 80 days post dosing. Total lymphocytes weremonitored for repopulation in the blood.

FIGS. 60A-60E show the level of CD4+ T cells, CD8+ T cells and B220+ Bcells in the blood 72 hours after dosing with 12G6-SFD1/K11 antibodies.FIGS. 61A-61E show the level of CD4+ T cells, CD8+ T cells and B220+ Bcells in the spleen 72 hours after dosing with 12G6-SFD1/K11 antibodies.FIGS. 62A-62F show the levels of circulating cytokines 2 hours afterdosing with 12G6-SFD1/K11 (“12G6 hu”) antibodies. FIG. 63 shows therepopulation of circulating lymphocytes over a timecourse after dosingwith 12G6-SFD1/K11 antibodies.

Example 48 Analysis of PK Profile of Anti-CD52 Antibodies in CD52Transgenic Mice (2C3-SFD1/K12, 12G6-SFD1/K11 and 9D9-H10/K12)

The pharmacokinetic profiles of anti-CD52 antibodies were determined inhuCD52 transgenic mice. This experiment compared the humanized andchimeric forms of the antibodies to ensure that the humanization processdid not alter the clearance rate of the antibodies. Comparisons includedchimeric 2C3, 12G6, and 9D9 antibodies and humanized 2C3-SFD1/K12,12G6-SFD1/K11, and 9D9-H10/K12 antibodies. Mice were injected i.v. withantibodies at 5 mg/kg and blood was collected at various timepointsbeginning two hours post dosing. The circulating levels of each antibodywere evaluated using an anti-human IgG ELISA. For each of thechimeric/humanized antibody pairs analyzed, there was a slightdifference in the Cmax noted at 2 hours post dosing. For the 2C3 and12G6 antibodies, the Cmaxof the humanized version (i.e., 2C3-SFD1/K12and 12G6-SFD1/K11) was slightly higher while the chimeric version wasslightly higher for the 9D9 pair. Clearance rates for the antibody pairswere similar over the course of the experiment indicating that thehumanization process did not significantly alter the pharmacokineticprofile of the antibodies.

FIGS. 64A-64C show the level of 2C3-chimeric, 2C3-SFD1/K12,12G6-chimeric, 12G6-SFD1/K11, 9D9-chimeric, and 9D9-H10/K12 antibodiesin the blood over a timecourse after dosing.

Example 49 Analysis of Depletion and Repopulation of Anti-CD52Antibodies in huCD52 Transgenic Mice (9D9-H10/K12)

The depleting activity of the 9D9-H10/K12 clone at different dose levelswas examined in the huCD52 transgenic mouse. Mice were injectedintravenously with 0.1, 0.5, 1.0 or 5.0 mg/kg of antibody. Two hourspost dosing, serum was collected to potentially examine the level ofcirculating cytokines. Three days post dosing, mice were sacrificed, andblood and spleens were collected from each mouse (N=5) to determine thelevel of cell depletion using flow cytometry analysis. Samples wereevaluated to determine the relative numbers of total T helper cell(CD4+), cytotoxic T cell (CD8+), B cell (B220+) and myeloid cellsubpopulations present in the circulating peripheral blood or spleen ofhuCD52 transgenic mice. In addition, T and B cell subset analysis wasperformed to determine the overall depleting effect. A subset of mice(N=5) were kept alive to monitor the repopulation kineticsAdministration of 9D9-H10/K12 at the 5, 1, and 0.5 mg/kg doses resultedin depletion of a significant number of both T cells and B cells in theblood. Only a modest level of lymphocyte depletion was observed in theblood at the 0.1 mg/kg dose. These data also demonstrated that various Tand B cell subsets are depleted to differing degrees depending on thedose of antibody used. Naïve T cells (both CD4 and CD8) demonstrated themost depletion compared to other cell populations (including memory andT reg cells), which were depleted to a lesser degree. In the B cellcompartment, mature B cells were depleted more readily than immature Bcells. In the spleen, significant depletion of these cells was onlyobserved at the 5 and 1 mg/kg dose levels. Similar to Campath-1H®, naïveT cells were more readily depleted than memory cells. Depletion wasobserved for NK cells and neutrophils in the blood but little to nodepletion was observed in the spleen at any of the doses injected. Serumcytokine analysis demonstrated no significant increases for either TNFαor IL-6 at any of the dose levels analyzed. Dose dependent increases inthe level of circulating MCP-1, however, were noted.

The repopulation portion of this experiment was terminated early whenlymphocytes were 50-80% repopulated (depending on the dose). Lymphocyterepopulation was monitored based on total lymphocyte counts and not on aT and B cell basis.

FIGS. 65A-65E show the level of CD4+ T cells, CD8+ T cells and B220+ Bcells in the blood 72 hours after dosing with 9D9-H10/K12 (“9D9”)antibodies. FIGS. 66A-66E show the level of CD4+ T cells, CD8+ T cellsand B220+ B cells in the spleen 72 hours after dosing with 9D9-H10/K12(“9D9”) antibodies. FIGS. 67A-67F show the levels of circulatingcytokines 2 hours after dosing with 9D9-H10/K12 (“9D9”) antibodies. FIG.68 shows the repopulation of circulating lymphocytes over a timecourseafter dosing with 9D9-H10/K12 (“9D9”) antibodies.

Example 50 Analysis of Depletion and Repopulation of Anti-CD52Antibodies in huCD52 Transgenic Mice (2C3-SFD1/K12, 12G6-SFD1/K11 and9D9-H10/K12)

The depleting activity of Campath-1H® and the humanized 2C3-SFD1/K12,12G6-SFD1/K11 and 9D9-H10/K12 clones at different dose levels wasexamined in the huCD52 transgenic mouse. Mice were injectedintravenously with either 0.1 or 1.0 mg/kg of antibody. Two hours postdosing, serum was collected to potentially examine the level ofcirculating cytokines. Three days post dosing, mice were sacrificed, andblood and spleens were collected from each mouse to determine the levelof cell depletion using flow cytometry analysis. Samples were evaluatedto determine the relative numbers of total T helper cell (CD4+),cytotoxic T cell (CD8+), B cell (B220+) and myeloid cell subpopulationspresent in the circulating peripheral blood or spleen of huCD52transgenic mice. In addition, T and B cell subset analysis was performedto determine the overall depleting effect. All of the humanizedantibodies (2C3-SFD1/K12, 12G6-SFD1/K11 and 9D9-H10/K12) mediateddepletion of lymphocytes within the spleen and blood when compared withPBS treated animals. Depletion was more robust in the blood than thespleen for all antibodies, and the depletion was dose-dependent in bothtissues. Depletion was most dramatic for CD4 and CD8+ T cells with lessdepletion in the B cell compartment. Various T and B cell subsets weredepleted to differing degrees. Naïve T cells (both CD4 and CD8)demonstrated the most depletion compared to other cell populations(including memory and T reg cells), which were depleted to a lesserdegree. In the B cell compartment, mature B cells were depleted morereadily than immature B cells. Serum cytokine analysis revealedsignificant increases in the level of IL-6, MCP-1 and TNFα 2 hours postdosing. Increases were noted for all antibodies, including Campath-1H®,and were dose dependent (i.e. higher cytokine levels were noted for the1.0 mg/kg dose level than the 0.1 mg/kg dose). In comparison toCampath-1H®, 2C3-SFD1/K12 and 12G6-SFD1/K11 induced similar levels ofIL-6 while 9D9-H10/K12 induced IL-6 to a significantly lower degree. ForMCP-1, the 12G6-SFD1/K11 antibody induced lower levels, and both12G6-SFD1/K11 and 9D9-H10/K12 decreased TNFα levels compared toCampath-1H®.

FIGS. 69A-69D show the level of bulk lymphocyte populations (CD4+ Tcells, CD8+ T cells, and B cells) and CD4+ T cell, CD8+ T cell and B220+B/NK cell subtypes in the blood 72 hours after dosing with Campath-1H®(“Campath”), 2C3-SFD1/K12 (“2C3”), 12G6-SFD1/K11 (“12G6”), and9D9-H10/K12 (“9D9”) antibodies. FIGS. 70A-70D show the level of bulklymphocyte populations (CD4+ T cells, CD8+ T cells, and B cells) andCD4+ T cell, CD8+ T cell and B220+ B/NK cell subtypes in the spleen 72hours after dosing with Campath-1H® (“Campath”), 2C3-SFD1/K12 (“2C3”),12G6-SFD1/K11 (“12G6”), and 9D9-H10/K12 (“9D9”) antibodies. FIGS.71A-71F show the levels of circulating cytokines 2 hours after dosingwith Campath-1H®, 2C3-SFD1/K12, 12G6-SFD1/K11, and 9D9-H10/K12antibodies.

Example 51 Direct Comparison of Anti-huCD52 Humanized 9D9 Clones inhuCD52 Transgenic Mice (9D9 H10/K12 and 9D9 H11/K12)

The depleting activity of two humanized anti-CD52 9D9 clones(9D9-H10/K12 and 9D9-H11/K12) was examined in huCD52 transgenic mice.Mice were injected intravenously with either 0.1 or 1.0 mg/kg ofantibody. Three days post dosing, mice were sacrificed, and blood andspleens were collected from each mouse to determine the level of celldepletion using flow cytometry analysis. Samples were evaluated todetermine the relative numbers of total T helper cell (CD4+), cytotoxicT cell (CD8+), B cell (B2200+) and NK cell subpopulations present in thecirculating peripheral blood or spleen of huCD52 transgenic mice.Treatment with either antibody resulted in similar lymphocyte depletionwithin the blood and spleen, with lymphocyte depletion in the bloodbeing more robust. Further, CD4 and CD8+ T cells were more stronglydepleted than B cells and NK cells in both tissues. While the depletionwith the 9D9-H10/K12 clone appears less robust than the depletion withthe 9D9-H11/K12 clone, the difference is not statistically significant.

FIG. 72 shows the level of CD4+ T cells, CD8+ T cells, B220+ B cells,and NK cells in the blood 72 hours after dosing with 9D9-H10/K12 and9D9-H11/K12 antibodies. FIG. 73 shows the level of CD4+ T cells, CD8+ Tcells, B220+ B cells, and NK cells in the spleen 72 hours after dosingwith 9D9-H10/K12 and 9D9-H11/K12 antibodies.

Example 52 Direct Comparison of Anti-huCD52 Humanized 12G6 Clones inhuCD52 Transgenic Mice (12G6-SFD1/K11 and 12G6-SFD1-K12)

The depleting activity of two humanized anti-CD52 12G6 clones(12G6-SFD1/K11 and 12G6-SFD1/K12) was examined in the huCD52 transgenicmouse. Mice were injected intravenously with either 0.1 or 1.0 mg/kg ofantibody. Two hours post dosing, serum was collected to potentiallyexamine the level of circulating cytokines. Three days post dosing, micewere sacrificed, and blood and spleens were collected from each mouse todetermine the level of cell depletion using flow cytometry analysis.Samples were evaluated to determine the relative numbers of total Thelper cell (CD4+), cytotoxic T cell (CD8+), B cell (B220+) and myeloidcell subpopulations present in the circulating peripheral blood orspleen of huCD52 transgenic mice. In addition, T and B cell subsetanalysis was performed to determine the overall depleting effect.Administration of either the 12G6-SFD1/K11 antibody or the 12G6-SFD1/K12antibody resulted in a significant level of lymphocyte depletion withinthe blood. There appeared to be little to no difference in thelymphocyte depleting activity of the two clones. The pattern oflymphocyte depletion was s such that naïve CD4 and CD8+ T cells weredepleted to a higher degree than memory T cells or Treg cells. Myeloidcell populations were depleted to a lesser degree regardless of theclone (12G6-SFD1/K11 or 12G6-SFD1/K12) or dose. Serum cytokine analysiswas not performed for this experiment.

FIGS. 74A-74D show the level of CD4+ T cells, CD8+ T cells, B220+ B/NKcells, and myeloid cells in the blood 72 hours after dosing with12G6-SFD1/K11 (“12G6 K11”) and 12G6-SFD1/K12 (“12G6 K12”) antibodies.FIGS. 75A-75D show the level of CD4+ T cells, CD8+ T cells, B220+ B/NKcells, and myeloid cells in the spleen 72 hours after dosing with12G6-SFD1/K11 (“12G6 K11”) and 12G6-SFD1/K12 (“12G6 K12”) antibodies.

Example 53 Direct Comparison of Anti-huCD52 Humanized 9D9 Clones inhuCD52 Transgenic Mice (9D9 H11/K12, 9D9 H16/K13, and 9D9 H18/K13)

The depleting activity of three humanized 9D9 antibodies (9D9-H11/K12,9D9-H16/K13, and 9D9-H18/K13) was compared in the huCD52 transgenicmouse. Human CD52 transgenic mice were treated with PBS as a vehiclecontrol or injected with either 1 mg/kg or 0.1 mg/kg of each antibody.At two hours post dosing, serum was collected to determine the level ofcirculating cytokines. Three days later, mice were sacrificed, andperipheral blood and spleens were collected and processed for flowcytometry analysis. Samples were evaluated to determine the relativenumbers of total T helper cell (CD4+), cytotoxic T cell (CD8+), B cell(B220+) and myeloid cell subpopulations present in the circulatingperipheral blood or spleen of huCD52 transgenic mice. In addition, T andB cell subset analysis was performed to determine the overall depletingeffect. All 9D9 (9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13) antibodiesmediated cellular depletion of lymphocyte and myeloid cell populationsin the blood and spleen to a similar extent. More robust lymphocyte andmyeloid cell depletion was observed in the blood than the spleen.Comparison of the depleting activity of the 9D9 clones (9D9-H11/K12,9D9-H16/K13, and 9D9-H18/K13) demonstrated that 9D9-H16/K13 resulted inthe most robust depletion, followed by 9D9-H18/K13 and 9D9-H11/K12. Thiswas most apparent for lymphocytes in the spleen at the 1 mg/kg dose inwhich 9D9-H16/K13 treatment resulted in a higher degree of depletionthan either of the other clones (9D9-H18/K13 and 9D9-H11/K12). Further,the pattern of depletion was such that naïve CD4 and CD8+ T cells weredepleted to a higher degree than memory T cells or Treg cells, and Bcell populations were depleted to a higher level with 9D9-H16/K13.Myeloid cell populations were less impacted by anti-CD52 treatmentregardless of the clone of antibody (9D9-H11/K12, 9D9-H16/K13, or9D9-H18/K13) or dose. Of the cytokines analyzed, increases were noted inIL-6, TNFα and MCP-1. Following injection, similar circulating level ofIL6 and MCP-1 were observed for all of the 9D9 clones (9D9-H11/K12,9D9-H16/K13, and 9D9-H18/K13) at both the 0.1 and 1.0 mg/kg dose levels.Slight differences were observed with circulating TNFα levels in whichinjection of the 9D9-H16/K13 clone resulted in a modest increase at the1.0 mg/kg dose.

FIG. 76 shows the level of bulk lymphocyte populations (CD4+ T cells,CD8+ T cells, and B220+ B cells) in the blood 72 hours after dosing with9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies. FIGS. 77A-77D showthe level of CD4+ T cell, CD8+ T cell, B220+ B/NK cell, and myeloid cellsubtypes in the blood 72 hours after dosing with 9D9-H11/K12,9D9-H16/K13, and 9D9-H18/K13 antibodies. FIG. 78 shows the level of bulklymphocyte populations (CD4+ T cells, CD8+ T cells, and B220+ B cells)in the spleen 72 hours after dosing with 9D9-H11/K12, 9D9-H16/K13, and9D9-H18/K13 antibodies. FIGS. 79A-79D show the level of CD4+ T cell,CD8+ T cell, B220+ B/NK cell, and myeloid cell subtypes in the spleen 72hours after dosing with 9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13antibodies. FIGS. 80A-80F show the levels of circulating cytokines 2hours after dosing with 9D9-H11/K12, 9D9-H16/K13, and 9D9-H18/K13antibodies.

Example 54 Analysis of PK Profile of Anti-CD52 Antibodies from the 2C3,12G6, and 9D9 Families in CD52 Transgenic Mice

The pharmacokinetic profiles of humanized 2C3-SFD1/K12, 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13 and 9D9-H18/K13 were determined in huCD52transgenic mice. Mice were injected i.v. with antibodies at 1 mg/kg andblood was collected at various timepoints beginning two hours postdosing. The circulating levels of each antibody were evaluated using ananti-human Ig ELISA. The calculated half-lives were: 2C3-SFD1/K1279.0±23.9 hours, 12G6-SFD1/K11 49.0±14.4 hours, 12G6-SFD1/K12 75.1±28.5,9D9-H16/K13 59.8+26.6 hours and 9D9-H18/K13 42.2+15.7 hours.

Overall, there was significant inter-animal variability for exposure inthese studies. The terminal elimination half-lives for 2C3-SFD1/K12 and12G6-SFD1/K12 were similar while the half-life of 12G6-SFD1/K11 wasshorter but not significantly different. Clearance was fastest with2C3-SFD1/K12 followed by 12G6-SFD1/K11 and 12G6-SFD1/K12. The two 12G6treatments mirrored each other for most of the time points measured,while 2C3-SFD1/K12 showed less exposure and faster clearance.9D9-H16/K13 and 9D9-H18/K13 were quite similar for all PK parametersmeasured.

FIGS. 81A-81B show the level of 2C3-SFD1/K12, 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13 and 9D9-H18/K13 antibodies in the blood overa timecourse after dosing.

TABLE 16 PK Parameters 2C3-SFD1/K12 12G6-SFD1/K11 12G6-SFD1/K129D9-H16/K13 9D9-H18/K13 t_(1/2) (hr) 79.0 ± 23.9 49.0 ± 14.4 75.1 ± 28.559.8 ± 26.6 42.2 ± 15.7 Cl (ml/hr/kg) 20.3 ± 2.9  10.6 ± 1.69 7.08 ±1.80 5.64 ± 1.73 6.65 ± 3.02 Vz (ml/kg) 2251 ± 539  770 ± 294 721 ± 224445 ± 133 366 ± 100 AUC (ug * hr/ml)  251 ± 37.2 485 ± 104 747 ± 188 196 ± 70.2  174 ± 65.2 Cmax (ug/ml) 4.22 ± 0.54 7.12 ± 1.97 8.96 ± 2.333.58 ± 2.16 4.35 ± 1.54

Example 55 Evaluation of Cytokine Storm in Response to Treatment withAnti-CD52 Antibodies

The release of serum cytokine following treatment with anti-CD52antibodies was evaluated in huCD52 transgenic mice. Animals were treatedwith 1 mg/kg of Campath-1H®, 12G6-SFD1/K11, 12G6-SFD1/K12, 9D9-H16/K13or 9D9-H18/K13. One group of animals was treated with 5 mg/kg of2C3-SFD1/K12 in view of previous results indicating that injection with2C3-SFD1/K12 may result in lower levels of depletion compared to theother antibodies, thereby normalizing the groups based on the doseneeded to achieve similar levels of depletion. All groups were bled 1 2,4, 24, and 48 hours post treatment and CBA analysis for inflammatorycytokines was conducted. All groups were also sacrificed 3 days posttreatment and the spleens were evaluated for depletion of lymphocytes inthe spleen by flow cytometry. Treatment with each of the antibodiesresulted in depletion of various targets similar to that observed forCampath-1H®. This was also true for 2C3-SFD1/K12, in which a 5 mg/kgdose was used to elicit similar depletion. Some variability in depletionwas observed with 12G6-SFD1/K12 and 9D9-H16/K13, most likely due to therepeated bleeding of the animals to acquire serum for cytokine analysis.Cytokine expression, however, was reduced for antibodies from the 12G6(12G6-SFD1/K11 and 12G6-SFD1/K12) and 9D9 (9D9-H16/K13 and 9D9-H18/K13)family members. This was most noticeable for release of IL-6, MCP-1 andTNFα at the early 1 and 2 hour time points.

FIGS. 82A-82F show the level of cytokines in the blood over a 48-hourtimecourse following dosing with Campath-1H® (“Campath”), 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13 or 9D9-H18/K13 antibodies. FIGS. 83A-83E showthe level of bulk lymphocytes, CD4+ T cells, CD8+ T cells, B220+ B/NKcells, and myeloid cells in the spleen 72 hours after dosing withCampath-1H® (“Campath”), 12G6-SFD1/K11, 12G6-SFD1/K12, 9D9-H16/K13 or9D9-H18/K13 antibodies.

Example 56 Evaluation of the Repopulation Kinetics in the Blood of CD52Transgenic Mice Following Treatment with Anti-CD52 Antibodies

The repopulation kinetics of several cell types in the blood wereassessed following administration of humanized anti-CD52 2C3-SFD1/K12,9D9-H16/K13 and 12G6-SFD1/K12 antibodies. Mice were injected i.v. witheach antibody at 2 mg/kg to ensure a robust level of depletion. Atvarious timepoints post injection, blood was collected for flowcytometry analysis to determine the level of circulating lymphocytes inthe blood, including CD4+ and CD8+ T cells, regulatory T cells, B cells,NK cells, neutrophils and macrophages. No differences were observed inthe initial depleting activity for each antibody, which was confirmed onday 3 post injection. Mice were bled weekly for the first month andbiweekly thereafter to monitor the kinetics of repopulation. Thekinetics of lymphocyte repopulation were similar for any of theanti-CD52 (2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12) antibodiescompared to Campath-1H®. By day 57, the B cells returned to baseline inthe blood while T cells approached baseline levels by day 84. By day116, CD8+ T cells had not returned to control levels, but similarrepopulation kinetics for all other cell types monitored were observedwith each of the anti-CD52 (2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12)antibodies and Campath-1H®.

FIGS. 84A-84G show the repopulation of circulating CD4+ and CD8+ Tcells, regulatory T cells, B cells, NK cells, neutrophils andmacrophages over a timecourse after dosing with Campath-1H® (“Campath”),2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12 antibodies.

Example 57 Evaluation of CD52 Expression in CD52 Transgenic Mice Usingthe Anti-CD52 Antibodies

Expression of huCD52 was evaluated using the humanized anti-CD52antibodies to determine whether similar staining patterns could beobserved on mature and developing cell populations in huCD52 transgenicmice. 2C3-SFD1/K12, 12G6-SFD1/K11, 12G6-SFD1/K12, 9D9-H16/K13, and9D9-H18/K13 antibodies were conjugated with FITC to use in flowcytometry staining. Tissues from huCD52 transgenic mice were collectedand processed for staining. 2C3-SFD1/K12, 12G6-SFD1/K11, 12G6-SFD1/K12,9D9-H16/K13, and 9D9-H18/K13 antibodies stained lymphocytes expressinghuCD52 from the spleen of transgenic mice similar to Campath-1H®. Thestaining patterns were representative of the lymphocyte populations andsubsets found in other lymphoid organs such as the thymus and bonemarrow.

FIG. 85 shows the ability of FITC-labeled Campath-1H®, 2C3-SFD1/K12,12G6-SFD1/K11, 12G6-SFD1/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies tospecifically bind huCD52 lymphocyte cell populations in the spleen.

Example 58 Direct Comparison of Single Dose Treatment with Anti-huCD52in huCD52 Transgenic Mice

The depleting activity of several humanized anti-CD52 antibodies(2C3-SFD1/K12, 12G6-SFD1/K11, 12G6-SFD1/K12, 9D9-H16/K13, and9D9-H18/K13) was compared in the huCD52 transgenic mouse. Mice wereinjected with antibodies i.v. at 1 mg/kg. At 2-hours post dosing, serumwas collected for cytokine analysis. Three days later mice weresacrificed and blood and spleen collected to compare the level oflymphocyte depletion. Significant levels of B and T cell depletion wereobserved for all of the anti-CD52 antibodies (2C3-SFD1/K12,12G6-SFD1/K11, 12G6-SFD1/K12, 9D9-H16/K13, and 9D9-H18/K13) and werecomparable to those observed following Campath-1H® administration.Subset analysis also revealed no significant differences in the level ofdepletion for each antibody (2C3-SFD1/K12, 12G6-SFD1/K11, 12G6-SFD1/K12,9D9-H16/K13, and 9D9-H18/K13) in either blood or spleen. Followinginjection of Campath-1H®, there was a marked increase in the circulatinglevels of both IL-6 and TNFα. Although injection of each of theanti-CD52 antibodies (2C3-SFD1/K12, 12G6-SFD1/K11, 12G6-SFD1/K12,9D9-H16/K13, and 9D9-H18/K13) resulted in a significant decrease in thelevel of TNFα compared to Campath-1H®, the levels of IL-6 were similar.

FIGS. 86A-86E show the level of bulk lymphocyte populations (CD4+ Tcells, CD8+ T cells, and B220+ B cells) and CD4+ T cell, CD8+ T cell,B220+ B/NK cell, and myeloid cell subtypes in the blood 72 hours afterdosing with Campath-1H® (“Campath”), 2C3-SFD1/K12, 12G6-SFD1/K11,12G6-SFD1/K12, 9D9-H16/K13, and 9D9-H18/K13 antibodies. FIGS. 87A-87Eshow the level of bulk lymphocyte populations (CD4+ T cells, CD8+ Tcells, and B220+ B cells) and CD4+ T cell, CD8+ T cell, B220+ B/NK cell,and myeloid cell subtypes in the spleen 72 hours after dosing withCampath-1H® (“Campath”), 2C3-SFD1/K12, 12G6-SFD1/K11, 12G6-SFD1/K12,9D9-H16/K13, and 9D9-H18/K13 antibodies. FIGS. 88A-88C show the levelsof circulating cytokines 2 hours after dosing with Campath-1H®(“Campath”), 2C3-SFD1/K12, 12G6-SFD1/K11, 12G6-SFD1/K12, 9D9-H16/K13,and 9D9-H18/K13 antibodies.

Example 59 In Depth Depletion of Lymphocytes in huCD52 Transgenic MiceFollowing Single Dose Treatment with Anti-huCD52 Antibodies

Extensive depletion analysis was performed in the huCD52 transgenicmouse using anti-CD52 2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12antibodies. Mice (N=4) were injected i.v. with a single dose of eachantibody at 1 mg/kg. Three days later, the mice were sacrificed, andblood, spleen, lymph nodes, and thymus were collected to compare thelevel of lymphocyte depletion using multi-color flow cytometry analysis.Significant levels of B and T cell depletion were observed for all ofthe anti-CD52 2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12 antibodies andwere comparable to those observed following Campath1H® administration ineach tissue examined. Subset analysis also revealed no significantdifferences in the level of depletion for each antibody in either bloodor spleen. Significant levels of lymphocyte depletion were also observedin the lymph nodes of mice. There did, however, appear to be somevariability in the activity of the antibody, especially when looking atthe central and effector memory T cell subset. Due to technical issuesregarding the LSR-II and the CD8 stain, the thymus could not beevaluated.

FIGS. 89A-89D show the level of CD4+ T cell, CD8+ T cell, B220+ B cell,and NK/myeloid cell subtypes in the blood 72 hours after dosing withCampath-1H® (“Campath”), 2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12antibodies. FIGS. 90A-90D show the level of CD4+ T cell, CD8+ T cell,B220+ B cell, and NK/myeloid cell subtypes in the spleen 72 hours afterdosing with Campath-1H® (“Campath”), 2C3-SFD1/K12, 9D9-H16/K13 and12G6-SFD1/K12 antibodies. FIGS. 91A-91D show the level of CD4+ T cell,CD8+ T cell, B220+ B cell, and NK/myeloid cell subtypes in the lymphnode 72 hours after dosing with Campath-1H® (“Campath”), 2C3-SFD1/K12,9D9-H16/K13 and 12G6-SFD1/K12 antibodies.

Example 60 Creation and Evaluation of the huCD52 Knock-In/Knock-Out(KI/KO) Transgenic Mouse on the C57BL/6 Background

A new human CD52 knock-in/knock-out mouse model was created on theC57Bl/6 background. To create this mouse, the mouse CD52 gene sequencewas replaced by the human CD52 gene sequence. The targeting strategyallowed for the replacement of the mouse sequence with the humansequence while maintaining the exon-intron structure. A selection markerwas used to identify progeny containing the new gene sequence. The finalallele was created by removal of the selection marker leaving only thehuman CD52 gene sequence.

Basic characterization of the huCD52 KI/KO mouse model involveddetermining the level of human CD52 expression on lymphocytes. Bloodfrom huCD52-KI/KO transgenic mice (N=4) and C57BL/6 mice (N=2) werestained for hCD52 expression and the number of CD52 molecules/cell wasenumerated using the Bang's labs Simply Cellular anti-human antibodyassay. Staining of peripheral blood cells from huCD52-KI/KO transgenicmice demonstrated that expression of huCD52 is very high on the majorityof lymphocytes from these animals. Expression levels were similar tothose observed in human CD4, CD8, and B cell populations. Expressionlevels on NK cells and macrophages were lower than those observed for Tcells and B cells. An increased level of huCD52 expression was detectedon neutrophils in these mice, contrary to the decreased expression levelin human neutrophils or similar cells from the original transgenic mouseline on the CD-1 background. Similar levels of CD52 expression wereobserved on T and B cells from the original huCD52 CD1 transgenic mouseand the huCD52 KI/KI mouse.

FIG. 92A shows the huCD52 expression level on CD4+ T cell, CD8+ T cell,B220+ B cell, and NK/myeloid cell subtypes in huCD52-KI/KO andnon-transgenic control mice. FIG. 92B shows the huCD52 expression levelon CD4+ T cells, CD8+ T cells, and B cells in huCD52-KI/KO and huCD52CD1 transgenic mice.

Example 61 Direct Comparison of Depletion Characteristics Between Smalland Large Scale Lots of 12G6 and 2C3

huCD52 KI/KO transgenic mice were dosed with 12G6-SFD1/K12 or2C3-SFD1/K12 to determine the depleting activity. In addition, activitywas examined using antibodies generated from two different sources(small scale and large scale lots) at Genzyme. Mice were injected i.v.with each antibody at 1 mg/kg. Three days post injection, mice weresacrificed, and blood was collected for flow cytometry analysis todetermine the levels of circulating CD4+ and CD8+ T cells, B cells, NKcells, neutrophils and macrophages. No significant differences indepletion of CD4 T cells, CD8+ T cells, B cells, and NK cells wereobserved between the small scale and large scale lot derived antibodies.

The various lots of 12G6-SFD1/K12 and 2C3-SFD1/K12 antibodies were alsoevaluated by flow cytometry to compare the intensity of staining onsplenocytes from huCD52-KI/KO transgenic mice. Both 12G6-SFD1/K12 and2C3-SFD1/K12 antibodies appear to recognize human CD52 to the sameextent as Campath-1H® on isolated splenocytes. In addition, there was nodifference in the level of recognition between the two sources (smallscale and large scale lots) of antibody.

FIGS. 93A-93B show binding to huCD52 of 12G6-SFD1/K12 and 2C3-SFD1/K12antibodies (from various production sources) as compared to aCampath-1H® control. FIG. 94 shows the level of bulk lymphocytepopulations (CD4+ T cells, CD8+ T cells, and B220+ B cells) in the blood72 hours after dosing with 12G6-SFD1/K12 and 2C3-SFD1/K12 antibodiesfrom various production sources.

Example 62 Analysis of PK Profile for Anti-CD52 Antibodies inhuCD52-KI/KO Transgenic Mice

The pharmacokinetic profiles of humanized 2C3-SFD1/K12, 9D9-H16/K13 and12G6-SFD1/K12 antibodies were determined in huCD52 KI/KO transgenicmice. Mice were injected i.v. with antibodies at 1 mg/kg, and blood wascollected at various timepoints beginning two hours post dosing. Thecirculating levels of each antibody were evaluated using an anti-humanIg ELISA. The overall clearance rate was similar for each of thehumanized anti-CD52 2C3-SFD1/K12, 9D9-H16/K13 and 12G6-SFD1/K12antibodies with 2C3-SFD1/K12 exhibiting potentially faster kinetics,while 12G6-SFD1/K12 was present in the serum for the longest period oftime.

FIGS. 95A-95B show the levels of 2C3-SFD1/K12, 9D9-H16/K13 and12G6-SFD1/K12 antibodies in the blood over a timecourse after dosing.

Example 63 Evaluation of 12G6 and 2C3 Pretreatment on EAE inhuCD52-KI/KO Transgenic Mice

The efficacy of anti-CD52 antibody treatment on reducing the overalldisease incidence and severity of Experimental AutoimmuneEncephalomyelitis (EAE) was evaluated in huCD52 KI/KO mice. huCD52-KI/KOmice were treated with a course of either 2C3-SFD1/K12 or 12G6-SFD1/K12on days −5 thru −1. EAE (a model of multiple sclerosis) was induced byimmunization with MOG35-55 peptide emulsified in CFA, and treatment withpertussis toxin, on days 0 and 2. Vehicle treated mice began to displaysigns of paralysis by day 10 post injection, which developed into severeprogressive disease. In contrast, pretreatment of mice with either the2C3-SFD1/K12 or 12G6-SFD1/K12 antibody delayed the onset of disease anddecreased the overall disease severity.

FIG. 96 demonstrates the EAE clinical score of 2C3-SFD1/K12 and12G6-SFD1/K12 over a timecourse of disease progression.

Example 64 Fc Modification of Antibodies to Alter the PharmacokineticProfile of Anti-CD52 Antibodies

Alterations in the Fc region of antibodies 1) affect the biologicalactivity of the antibody by altering interactions with Fc receptorsand/or 2) alter the pharmacokinetic profile of the antibody by alteringinteractions with the FcRn neonatal receptor. The FcRn molecule isexpressed on vascular endothelium and is believed to be the main site ofIgG recycling. The FcRn binds to the antibody Fc portion which thenbecomes internalized within a cell. Antibodies that have high affinityinteractions with the FcRn will be recycled back to the surface of thecell and will be released back into circulation. Antibodies that havelower affinity interactions dissociate within the cell and ultimatelydegrade. Site directed mutagenesis to increase the interaction with FcRngenerates an antibody that can be maintained in circulation for longerperiods of time compared to an unmodified antibody. Conversely,mutations within the Fc region of an antibody that decrease FcRn bindingshorten the circulating half-life of the antibody. Mutations that havebeen described to decrease binding to FcRn resulting in shortercirculating half-lives include the His435Ala single mutation and theHis310Ala/His435Gln double mutation (see, e.g., Kim et al., “Mapping thesite on human IgG for binding of the MHC class I-related receptor,FcRn,” Eur. J. Immunol., 29:2819-2825 (1999) and Kenanova et al.,“Tailoring the Pharmacokinetics and Positron Emission Tomography ImagingProperties of Anti-Carcinoembryonic Antigen Single-Chain Fv-Fc AntibodyFragments,” Cancer. Res. 65(2):622-631 (2005)).

The 2C3-SFD1/K12 antibody was mutated to generate His435Ala 2C3-SFD1/K12(“2C3-SFD1/K12-Modified 1”) and His310Ala/His435Gln 2C3-SFD1/K12(“2C3-SFD1/K12-Modified 2”) antibodies that have altered PK profiles.Biacore analysis was conducted to confirm decreased binding to bothmouse and human FcRn molecules. Both Campath-1H® and 2C3-SFD1/K12antibodies bound to each of the mouse and human FcRn molecules withsimilar kinetics. In contrast, His435Ala 2C3-SFD1/K12 antibodies boundat low levels to the mouse FcRn but not to human FcRn.His310Ala/His435Gln 2C3-SFD1/K12 antibodies did not bind to either mouseor human FcRn molecule, indicating that the incorporation of either thesingle or double mutation into the 2C3-SFD1/K12 Fc region significantlyaffects binding to mouse and human FcRn.

FIGS. 97A-97B demonstrate the ability of Campath1H® (“Campath”),2C3-SFD1/K12 (“2C3”), His435Ala 2C3-SFD1/K12 (“H435A 2C3”) andHis310Ala/His435Gln 2C3-SFD1/K12 (“H310A/H435Q 2C3”) to bind to mouseand human FcRn molecules.

Example 65 Evaluation of the Half-Life of Fc Modified Anti-CD52Antibodies Following I.V. Administration in C57Bl/6 Mice

Fc modifications were incorporated into the 2C3-SFD1/K12 backbone togenerate 2C3-SFD1/K12-Modified 1 and 2C3-SFD1/K12-Modified 2 antibodiesthat exhibited decreased binding to the FcRn receptor responsible formaintaining antibodies in circulation. The pharmacokinetic profile wasdetermined for the 2C3-SFD1/K12 antibody and the 2C3-SFD1/K12-Modified 1and 2C3-SFD1/K12-Modified 2 antibodies with reduced FcRn binding.C57BL/6 mice were used to evaluate the PK profile in the absence oftarget antigen (2C3-SFD1/K12 binds to human CD52 but does notcross-react with mouse CD52). Mice were injected i.v. with antibodies at1 mg/kg. At various timepoints, blood was collected to analyze the levelof circulating human IgG1 in the mouse serum by ELISA. Both2C3-SFD1/K12-Modified 1 and 2C3-SFD1/K12-Modified 2 antibodies werecleared from the blood faster than the 2C3-SFD1/K12 antibody.2C3-SFD1/K12 had a half-life of 403 hrs, while 2C3-SFD1/K12-Modified 1had a half-life of 51 hours and 2C3-SFD1/K12-Modified 2 had a half-lifeof 8 hours. PK profiles for 2C3-SFD1/K12 and 2C3-SFD1/K12-Modified-1were consistent with a 1-compartment model with only a single phase ofelimination. In contrast, profiles for 2C3-SFD1/K12-Modified-2 wereconsistent with a 2 compartment model, with 2 distinct phases ofelimination (specified as alpha and beta in the table). The first phaselasted until 48 hr post dose (alpha) and the second phase (beta, alsocalled the terminal elimination phase) started 48 hr post dose.

FIG. 98 shows the in vivo clearance of 2C3-SFD1/K12 (“2C3 unmodified”),2C3-SFD1/K12-Modified 1 (“2C3-Fc mutant 1”) and 2C3-SFD1/K12-Modified 2(“2C3-Fc mutant 2”) in nontransgenic mice.

TABLE 17 Summary of Pharmacokinetic Data Across Groups 2C3-SFD1/K12-2C3-SFD1/K12- 2C3-SFD1/K12 Modified 1 Modified 2 t_(1/2) (hr) 403 ± 14051.0 ± 12.3 8.05 ± 0.74 (Alpha) 282 ± 385 (Beta) Cl (ml/hr/kg) 0.29 ±0.09 1.35 ± 0.36 5.90 ± 4.67 Vz (ml/kg)  156 ± 40.7 94.8 ± 14.3 1932 ±1341 AUC (ug * hr/ml) 3748 ± 937  781 ± 171 230 ± 105 Cmax (ug/ml) 9.65± 1.72 11.9 ± 0.83 9.64 ± 3.70

TABLE 18 Individual Animal Data HL_Lambda_z Cmax AUCINF_obs Vz_obsCl_obs Group^(#) Animal (hr) (ug/ml) (hr * ug/ml) (ml/kg) (ml/hr/kg) 2C32.1 197.26 11.56 2967.86 95.89 0.34 2C3 2.2 494.01 10.54 4635.96 153.730.22 2C3 2.3 324.61 10.06 3783.76 123.77 0.26 2C3 2.4 283.68 10.573130.92 130.72 0.32 2C3 2.5 330.89 6.15 2025.29 235.71 0.49 2C3 2.6547.78 10.56 4469.73 176.81 0.22 2C3 2.7 597.92 10.57 4764.75 181.040.21 2C3 2.8 320.65 7.61 3415.82 135.43 0.29 2C3 2.9 527.01 9.27 4533.82167.70 0.22 AVG 402.65 9.65 3747.55 155.64 0.29 SD 140.22 1.72 937.3840.73 0.09 2C3-M1 3.1 35.20 12.84 513.50 98.89 1.95 2C3-M1 3.2 42.7411.68 842.55 73.17 1.19 2C3-M1 3.3 50.55 11.39 902.62 80.80 1.11 2C3-M13.4 46.61 12.49 717.95 93.67 1.39 2C3-M1 3.5 56.38 12.94 911.32 89.261.10 2C3-M1 3.6 63.40 12.41 995.22 91.91 1.00 2C3-M1 3.7 33.86 12.02513.17 95.19 1.95 2C3-M1 3.8 63.14 10.56 842.79 108.08 1.19 2C3-M1 3.966.75 11.02 788.59 122.12 1.27 AVG 50.96 11.93 780.86 94.79 1.35 SD12.30 0.83 170.51 14.33 0.36 Alpha Beta HL_Lambda HL_Lambda CmaxAUCINF_obs Vz_obs Cl_obs Group^(#) Animal (hr) (hr) (ug/ml) (hr * ug/ml)(ml/kg) (ml/hr/kg) 2C3-M2 4.1* 8.31 Missing 10.62 177.07  67.74 5.652C3-M2 4.2 7.42 994.71 11.35 390.03 3679.37 2.56 2C3-M2 4.3 7.37 703.0910.48 315.82 3211.80 3.17 2C3-M2 4.4 7.72 227.03 11.78 247.96 1320.924.03 2C3-M2 4.5** Missing Missing Missing Missing Missing Missing 2C3-M24.6** Missing Missing Missing Missing Missing Missing 2C3-M2 4.7***77.89   77.89  1.32  61.71 1820.87 16.20  2C3-M2 4.8 8.18 150.98 11.41221.89  981.64 4.51 2C3-M2 4.9 9.33  77.61 10.49 194.31  576.21 5.15 AVG8.05 281.98  9.64 229.83 1931.80 5.90 SD 0.74 384.82  3.70 104.691341.43 4.67 ^(#)The tested groups were 2C3-SFD1/K12 (“2C3”),2C3-SFD1/K12-Modified 1 (“2C3-M1”) and 2C3-SFD1/K12-Modified 2(“2C3-M2”) *Animal 4.1 no beta t½, Vz outlier. **Animals 4.5 & 4.6, notenough data for PK analysis. ***Animal 4.7 incomplete injection

Example 66 Evaluation of the Half-Life of Fc Modified Anti-CD52Antibodies Following I.V. Administration in Heterozygous huCD52Transgenic Mice

The pharmacokinetic profile was determined for the 2C3-SFD1/K12 antibodyand the 2C3-SFD1/K12-Modified 1 and 2C3-SFD1/K12-Modified 2 antibodieswith reduced FcRn binding in vitro. huCD52 transgenic mice were used toevaluate the PK profile in the presence of the 2C3-SFD1/K12 antibodytarget antigen. Mice were injected i.v. with antibodies at 1 mg/kg. Atvarious timepoints, blood was collected to determine the level ofcirculating human IgG1 in the mouse serum by ELISA. Both2C3-SFD1/K12-Modified 1 and 2C3-SFD1/K12-Modified 2 antibodies werecleared from the blood faster than the 2C3-SFD1/K12 antibody.2C3-SFD1/K12 had a half-life of 64 hrs, while 2C3-SFD1/K12-Modified 1had a half-life of 32 hours, and 2C3-SFD1/K12-Modified 2 had a half-lifeof 6.5 hours.

FIG. 99 shows the in vivo clearance of 2C3-SFD1/K12 (“2C3”),2C3-SFD1/K12 modified 1 (“2C3-Fc mutant 1”) and 2C3-SFD1/K12 modified 2(“2C3-Fc mutant 2”) in huCD52 transgenic mice.

TABLE 19 Summary of Pharmacokinetic Data Across Groups 2C3-SFD1/K12-2C3-SFD1/K12- 2C3-SFD1/K12 Modified 1 Modified 2 t_(1/2) (hr) 64.2 ±12.1 32.3 ± 3.25 6.58 ± 2.03 Cl (ml/hr/kg) 2.15 ± 0.31 2.51 ± 0.28 5.41± 0.83 Vz (ml/kg)  198 ± 42.8  117 ± 21.1 49.7 ± 11.1 AUC (ug * hr/ml) 475 ± 73.4  403 ± 44.5  188 ± 27.2 Cmax (ug/ml) 8.88 ± 1.69 12.4 ± 1.6712.9 ± 1.91

TABLE 20 Individual Animal Data HL_Lambda_z Cmax AUCINF_obs Vz_obsCl_obs Group# Animal (hr) (ug/ml) (hr * ug/ml) (ml/kg) (ml/hr/kg) 2C32.1 77.32 8.19 421.87 264.42 2.37 2C3 2.11 61.47 10.38 483.25 183.512.07 2C3 2.2 78.28 9.28 496.58 227.42 2.01 2C3 2.3 82.38 6.99 441.98268.91 2.26 2C3 2.4 53.60 9.08 465.09 166.28 2.15 2C3 2.5 58.02 9.09526.59 158.95 1.90 2C3 2.6 44.97 6.03 371.17 174.78 2.69 2C3 2.7 56.529.38 476.41 171.16 2.10 2C3 2.8 67.99 12.13 641.28 152.97 1.56 2C3 2.961.46 8.30 421.65 210.29 2.37 Mean 64.20 8.88 474.59 197.87 2.15 SD12.06 1.69 73.40 42.80 0.31 2C3-M1 3.1 28.48 15.19 412.41 99.64 2.422C3-M1 3.11 34.64 12.60 468.36 106.69 2.14 2C3-M1 3.2 27.57 14.17 411.8296.60 2.43 2C3-M1 3.3 34.27 11.96 401.38 123.20 2.49 2C3-M1 3.4 29.1012.51 400.36 104.85 2.50 2C3-M1 3.5 29.63 11.11 470.98 90.77 2.12 2C3-M13.6 32.76 9.75 348.72 135.53 2.87 2C3-M1 3.7 35.68 10.41 328.71 156.613.04 2C3-M1 3.8 36.41 13.52 390.24 134.61 2.56 2C3-M1 3.9 34.06 12.39392.53 125.19 2.55 Mean 32.26 12.36 402.55 117.37 2.51 SD 3.25 1.6744.48 21.05 0.28 2C3-M2 4.1 7.64 13.45 197.24 55.85 5.07 2C3-M2 4.11Missing 9.00 Missing Missing Missing 2C3-M2 4.2 7.79 14.92 217.80 51.614.59 2C3-M2 4.3 7.35 12.79 183.44 57.78 5.45 2C3-M2 4.4 3.54 10.34152.92 33.44 6.54 2C3-M2 4.5 Missing Missing Missing Missing Missing2C3-M2 4.6 Missing Missing Missing Missing Missing 2C3-M2 4.7 MissingMissing Missing Missing Missing 2C3-M2 4.8 Missing Missing MissingMissing Missing 2C3-M2 4.9 Missing Missing Missing Missing Missing Mean6.58 12.10 187.85 49.67 5.41 SD 2.03 2.40 27.23 11.12 0.83 PK parametersnot available for 4.11, 4.5, 4.6, 4.7, 4.8, and 4.9 due to insufficientdata. #The tested groups were 2C3-SFD1/K12 (“2C3”),2C3-SFD1/K12-Modified 1 (“2C3-M1”) and 2C3-SFD1/K12-Modified 2(“2C3-M2”)

Example 67 Evaluation of In Vivo Depletion Following I.V. Administrationof Fc Modified Anti-CD52 Antibodies in Heterozygous huCD52 TransgenicMice

The depletion activity was determined for the 2C3-SFD1/K12,2C3-SFD1/K12-Modified 1, and 2C3-SFD1/K12-Modified 2 antibodies inhuCD52 transgenic mice. Mice were treated with 1 mg/kg of 2C3-SFD1/K12,2C3-SFD1/K12-Modified 1, or 2C3-SFD1/K12-Modified 2 antibodies andevaluated for the presence of CD4 T cells, CD8+ T cells, B cells, and NKcells 72 hours later. Administration of 2C3-SFD1/K12-Modified 1 or2C3-SFD1/K12-Modified 2 antibodies resulted in decreased levels ofdepletion in the blood and spleen compared administration of2C3-SFD1/K12 antibodies. Further, 2C3-SFD1/K12-Modified 1 elicitedgreater depletion than 2C3-SFD1/K12-Modified 2 in both the blood andspleen

FIGS. 100A-100B show the level of bulk lymphocyte populations (CD4+ Tcells, CD8+ T cells, B220+ B cells, and NK cells) in the blood andspleen 72 hours after dosing with 2C3-SFD1/K12 (“2C3”), 2C3-SFD1/K12modified 1 (“2C3 Fc mutant-1”), and 2C3-SFD1/K12 modified 2 (“2C3 Fcmutant-2”) antibodies.

Example 68 Detailed Epitope Specificities of Humanized Anti-CD52Antibodies

Detailed epitope specificities of the humanized 12G6-SFD1/K12,2C3-SFD1/K12, and 9D9-H16/K13 antibodies were determined using a BiacoreT100 instrument. As a control, the epitope specificity of clone 097(purified anti-human CD52 antibody, Biolegend) was evaluated using thesame methodologies. The epitope specificity of clone 097 had previouslybeen characterized using a peptide ELISA method (Hale G, “Syntheticpeptide mimotype of the CAMPATH-1 (CD52) antigen, a smallglycosylphosphatidylinositol-anchored glycoprotein,” Immunotechnology,1:175-187 (1995)). In this Biacore T100 assay, the antibodies weredirectly immobilized on Biacore CM5 Series S carboxymethyl dextransensor chips (GE #BR-1006-68) using amine coupling. The carboxymethyldextran surface was activated using a 1:1 mixture of 0.1MN-hydroxysuccinimide (NHS) and 0.4MN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC),allowing the surface to bind reactive amine groups on the antibodies.Because IgM antibodies tend to have a higher level of non-specificbinding compared to IgGs, the binding of a mouse IgMκ (mIgMκ) isotypecontrol (Biolegend clone #MM-30) was also investigated. Followingantibody immobilization, the reactive sensor chip surface was quenchedusing 1M ethanolamine hydrochloride/NaOH pH 8.5. One flow cell on eachchip was a blank reference surface, and subsequent flow cells wereimmobilized with 10,000 RU of antibody.

A series of alanine-scanning mutant peptides comprising the human CD52sequence (MUT 1-MUT 12 (SEQ ID NOS: 169-180, respectively), Table 21)(see, e.g., Hale G, “Synthetic peptide mimotype of the CAMPATH-1 (CD52)antigen, a small glycosylphosphatidylinositol-anchored glycoprotein,”Immunotechnology, 1:175-187 (1995)) were synthesized. Antibody bindingto these mutant CD52 peptides and to wildtype human CD52 peptides wastested at concentrations of 500 nM, 100 nM, 50 nM, and 0 nM. Peptideswere diluted into the assay running buffer, HBS-EP+ (10 mM HEPES, 150 mMNaCl, 0.05% P20 surfactant, 3 mM EDTA, pH 7.4). Duplicates of 100 nMsamples were included. The light (kappa) chain specific rat anti-mouseIgM antibody (Southern Biotech Clone #1B4B1) was also included as an IgMcontrol. The T100 instrument sample chamber and assay temperatures wereset to 4° C. and 25° C., respectively. The human CD52 peptide sampleswere injected for five minutes at a 50 μl/min flow rate to measureassociation, and washed in HBS-EP+ for five minutes at a 50 μl/min flowrate to measure dissociation. The antibody surface was stripped of anyremaining bound peptide using a sixty second injection of 10 mMglycine-HCl pH 2.0 at a 50 μl/min flow rate. Analysis was performedusing Biacore T100 Kinetics Evaluation software v2.0 (GE Healthcare).Data was fit to a 1:1 model with reference flow cell and 0 nMconcentration subtraction (double-reference subtraction). Representativesensorgrams of 12G6-SFD1/K12 antibody negative ((−), MUT 8) and positive((+), MUT 9) peptide epitope recognition are shown in FIG. 101A and FIG.101B, respectively. The compiled peptide binding data is summarized inTable 21.

The previously characterized binding specificity of clone 097 (Hale G,“Synthetic peptide mimotype of the CAMPATH-1 (CD52) antigen, a smallglycosylphosphatidylinositol-anchored glycoprotein,” Immunotechnology,1:175-187 (1995)) was determined by coating ELISA plates with peptidescontaining the six residues of the C-terminal portion of human CD52 andthen measuring the binding of the antibody to the fixed peptide. Each ofthe residues was substituted by all 20 amino acids. Because the peptideswere attached to a solid surface in this ELISA, the assay may have beenmore influenced by avidity effects than the Biacore T100 assay describedherein, which uses an antibody fixed to the surface over which thepeptides are flowed. In the ELISA study, alanine substitutions atpositions 11 and 12 (wildtype residues proline and serine, respectively)of the mature form of human CD52 were found to reduce strong binding ofclone 097 to the peptide. In the present Biacore T100 study, alaninesubstitutions at positions 11 and 12 (as well as positions 7, 8, 9, and10) were found to abrogate binding of clone 097. The hypothesizedavidity effects of the ELISA assay are likely the reason why the mappedepitope of clone 097 is smaller as determined by the ELISA method thanas determined by the described Biacore T100 assay.

The binding of both the 2C3-SFD1/K12 and 12G6-SFD1/K12 humanizedantibodies to the human CD52 peptide sequence is sensitive to alaninesubstitutions at positions 7, 8, and 11 and the binding of humanized9D9-H16/K13 is sensitive to alanine substitutions at positions 4 and 11.These defined epitope specificities overlap with the results observed inExample 4 (summarized in Table 8). Slight variations between the resultsare not unexpected given that the Biacore T100 method used to measurebinding in the present case was significantly different from the methodused in Example 4. In contrast to the present case, in Example 4,engineered CHO cells were used to express wildtype oralanine-substituted mutants of human CD52. Human CD52 expressed in suchmammalian cells can be glycosylated, affecting binding. This is not thecase for the human CD52 used in the Biacore T100 assay.

TABLE 21 Binding to alanine-scanning mutant hCD52 peptides 2C3- 9D9-12G6- Control SEQ SFD1/K12 H16/K13 SFD1/K12 097 mIgM Peptide ID NO:Peptide Sequence Binding Binding Binding Binding Binding MUT 1 169 AQNDTSQTSSPSADC + + + + − MUT 2 170 G A NDTSQTSSPSADC + + + + − MUT 3 171GQ A DTSQTSSPSADC + + + + − MUT 4 172 GQN A TSQTSSPSADC + − + + − MUT 5173 GQND A SQTSSPSADC + + + + − MUT 6 174 GQNDT A QTSSPSADC + + + + −MUT 7 175 GQNDTS A TSSPSADC − + − − − MUT 8 176 GQNDTSQ A SSPSADC − + −− − MUT 9 177 GQNDTSQT A SPSADC + + + − − MUT 10 178 GQNDTSQTS APSADC + + + − − MUT 11 179 GQNDTSQTSS A SADC − − − − − MUT 12 180GQNDTSQTSSP A ADC + + + − − Controls WT 1 181 GQNDTSQTSSPSADK- + + + + −Biotin WT 2 182 Biotin- + − + + − GQNDTSQTSSPSAD Rat anti-mIgM N/A N/AN/A N/A N/A + + (+) Binding detected: Maximum response (R_(max)) > 2RUsfor 500 nM peptide injection (−) No binding detected: Maximum response(R_(max)) < 2RUs for 500 nM peptide injection

Example 69 Assessment of CD4+ T Cell Responses Induced by Campath-1H® or12G6-SFD1/K12

The CD4+ T cell proliferative response was evaluated after repeated invitro stimulation with autologous dendritic cells (DC) preloaded with aset of overlapping 15-mer peptides comprising sequences from thevariable regions of either Campath-1H® or the humanized 12G6-SFD1/K12antibody. These experiments utilized normal human donor T cells and DCs.Results were measured by quantifying tritiated thymidine incorporationof the proliferating human CD4+ T cells in response to autologouspeptide pulsed antigen presenting cells (APC).

Cell preparation: PBMCs were isolated from a normal human donorapheresis product acquired from BioMed Supplies (Carlsbad, Calif.). HLAhaplotype screening of the donor blood was performed by Key Biologics,LLC (Memphis, Tenn.) (Table 22). PBMCs were isolated using theFicoll-Paque PLUS density gradient (GE Healthcare) and a series ofwashes with phosphate buffered saline (PBS, Invitrogen, Carlsbad,Calif.). CD4+ T cells were isolated from PBMC using the Dynal CD4+bead-based positive isolation kit (Invitrogen), following themanufacturer's recommended protocol. Isolated CD4+ T cells were frozenin Recovery Cell Culture Freezing Media (Invitrogen) and stored inliquid nitrogen. Dendritic Cells (DC) were induced from PBMCs by platingadherent cells with GM-CSF (Leukine, Bayer, Leverkusin, Germany) andIL-4 (Peprotech, Rocky Hill, N.J.) for six days. Media supplemented withGM-CSF and IL-4 was replaced on day 4. DCs were subsequently isolatedfrom the flasks and frozen in the Freezing Media then transferred toliquid nitrogen storage tanks.

TABLE 22 HLA haplotype of blood donors Donor HLA DR haploytpe peptideset BMS170 DRB1_0701 DRB1_1503 Campath BMS154 DRB1_0301 DRB1_0302Campath BMS150 DRB1_1101 DRB1_1302 Campath BMS167 DRB1_0701 DRB1_1503Campath BMS200 DRB1_0804 DRB1_1202 Campath BMS301 DRB1_1401 DRB1_1503Campath BMS352 DRB1_0301 DRB1_1101 Campath BMS362 DRB1_0302 DRB1_0302Campath BMS484 DRB1_0103 DRB1_1201 Campath/GLD52 BMS486 DRB1_1302DRB1_1303 Campath/GLD52 BMS640 DRB1_0301 DRB1_1302 GLD52 BMS656 DRB1_301DRB1_1101 GLD52 BMS902 DRB1_0302 DRB1_0804 GLD52 BMS928 DRB1_1001DRB1_1503 GLD52 BMS927 DRB1_1001 DRB1_1503 GLD52 BMS963 DRB1_0302DRB1_1401 GLD52 BMS361 DRB1_1102 DRB1_1401 GLD52 BMS165 DRB1_1102DRB1_1501 GLD52

Peptide: Peptides encompassing the heavy and light chain variableregions of Campath-1H® and 12G6-SFD1/K12 were synthesized using a RaininSymphony automated peptide synthesizer using standard Fmoc-chemistry onCLEAR resin (Peptides International, Louisville, Ky.) Amino acids (EMDBiosciences, San Diego, Calif. or Anaspec, San Jose, Calif.) wereorthogonally protected with tert-Butoxycarbonyl (BOC), tert-Butyl (tBu),2,2,4,6,7-Pentamethyldihydro-benzofuran-5-sulfonyl (Pbf), or Trityl(Trt) groups. Couplings were performed using an aminoacid/HCTU/HOBt/DIEA/resin with a molar ratio of 6:6:3:12:1. A solutionof 20% Piperidine and 2.5% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) inDMF was used to remove Fmoc from the amino terminus during each cycle.Deprotection/cleavage from resin was performed using a cocktail of 15mls/0.1 mM resin of 2.5% water/2.5% TIS/5% Anisole/90% TFA v/v ratio for3 hours. Supernatant was precipitated in diethyl-ether (−80° C.) andpelleted at 3000 rpm for 10 minutes. Ether was decanted and the pelletwas washed again. Crude peptide was then lyophilized. Analytical HPLC(XBridge C18 4.5×100 mm, Waters Corp., Milford, Mass.) and MALDI-TOFmass spectrometry (Synapt, Waters Corp., Milford, Mass.) were used toverify the sequences and assess purity. All reagents were HPLC grade(EMD Biosciences, San Diego, Calif. or Sigma Aldrich, St. Louis, Mo.).Lyophilized peptides were resuspended in 100% DMSO (Sigma). Forty threeCampath-1H® peptides were combined into 11 linear groups, eachcontaining 3 or 4 peptides per group (Table 23: from top to bottom,light chain peptides are denoted by SEQ ID NOs: 187-206 and heavy chainpeptides are denoted by SEQ ID NOs: 207-229). The 42 12G6-SFD1/K12peptides were combined into 8 linear groups, each containing five or sixpeptides per group (Table 24: from top to bottom, light chain peptidesare denoted by SEQ ID NOs: 230-250 and heavy chain peptides are denotedby SEQ ID NOs: 251-271).

TABLE 23 Campath-1H ® 15-mer light chain and heavy chainpeptides, overlapping by 10 amino acids each Campath-1H ®  Peptideslight chain heavy chain Peptide ID# Peptide ID# DIQMTQSPSSLSASV 978QVQLQESGPGLVRPS  998 QSPSSLSASVGDRVT 979 ESGPGLVRPSQTLSL  999LSASVGDRVTITCKA 980 LVRPSQTLSLTCTVS 1000 GDRVTITCKASQNID 981QTLSLTCTVSGFTFT 1001 ITCKASQNIDKYLNW 982 TCTVSGFTFTDFYMN 1002SQNIDKYLNWYQQKP 983 GFTFTDFYMNWVRQP 1003 KYLNWYQQKPGKAPK 984DFYMNWVRQPPGRGL 1004 YQQKPGKAPKLLIYN 985 WVRQPPGRGLEWIGF 1005GKAPKLLIYNTNNLQ 986 PGRGLEWIGFIRDKA 1006 LLIYNTNNLQTGVPS 987EWIGFIRDKAKGYTT 1007 TNNLQTGVPSRFSGS 988 IRDKAKGYTTEYNPS 1008TGVPSRFSGSGSGTD 989 KGYTTEYNPSVKGRV 1009 RFSGSGSGTDFTFTI 990EYNPSVKGRVTMLVD 1010 GSGTDFTFTISSLQP 991 VKGRVTMLVDTSKNQ 1011FTFTISSLQPEDIAT 992 TMLVDTSKNQFSLRL 1012 SSLQPEDIATYYCLQ 993TSKNQFSLRLSSVTA 1013 EDIATYYCLQHISRP 994 FSLRLSSVTAADTAV 1014YYCLQHISRPRTFGQ 995 SSVTAADTAVYYCAR 1015 HISRPRTFGQGTKVE 996ADTAVYYCAREGHTA 1016 RTFGQGTKVEIKRTV 997 YYCAREGHTAAPFDY 1017EGHTAAPFDYWGQGS 1018 APFDYWGQGSLVTVS 1019 WGQGSLVTVSSASTK 1020

TABLE 24 42 12G6-SFD1/K12 15-mer light chain andheavy chain peptides, overlapping by 10 amino acids each12G6-SFD1/K12 Peptides light chain heavy chain Peptide ID# Peptide ID#DIVMTQTPLSLSVTP 1027 EVQLVESGGGLVQPG 1048 QTPLSLVTPGQPAS 1028ESGGGLVQPGGSLRL 1049 LSVTPGQPASISCKS 1029 LVQPGGSLRLSCAAS 1050GQPASISCKSSQSLL 1030 GSLRLSCAASGFPFS 1079 ISCKSSQSLLYSNGK 1031SCAASGFPFSNYWMN 1080 SQSLLYSNGKTYLNW 1032 GFPFSNYWMNWVRQA 1081YSNGKTYLNWVLQKP 1072 NYWMNWVRQAPGKGL 1082 TYLNWVLQKPGQSPQ 1073WVRQAPGKGLEWVGQ 1055 VLQKPGQSPQRLIYL 1074 PGKGLEWVGQIRLKS 1056GQSPQRLIYLVSKLD 1036 EWVGQIRLKSNNYAT 1060 RLIYLVSKLDSGVPD 1037IRLKSNNYATHYAES 1061 VSKLDSGVPDRFSGS 1038 NNYATHYAESVKGRF 1062SGVPDRFSGSGSGTD 1039 HYAESVKGRFTISRD 1063 RFSGSGSGTDFTLKI 1040VKGRFTISRDDSKNS 1064 GSGTDFTLKISRVEA 1041 TISRDDSKNSLYLQM 1065FTLKISRVEAEDVGV 1042 DSKNSLYLQMNSLKT 1066 SRVEAEDVGVYYCVQ 1043LYLQMNSLKTEDTAV 1067 EDVGVYYCVQGSHFH 1075 NSLKTEDTAVYYCTP 1068YYCVQGSHFHTFGQG 1076 EDTAVYYCTPIDYWG 1083 GSHFHTFGQGTKLEI 1077YYCTPIDYWGQGTTV 1084 TFGQGTKLEIKRTVA 1078 IDYWGQGTTVTVSSA 1085In Vitro Stimulation

DC antigen pulsing and maturation: Before treatment with the peptides,DCs were thawed, washed and plated in RPMI (Invitrogen, Carlsbad,Calif.) supplemented with 5% Human Serum (HS, Sigma, St. Louis, Mo.), 1%Penicillin-Streptomycin (Invitrogen, Carlsbad, Calif.), 100 ng/mlGM-CSF, and 20 ng/ml IL-4. DCs were plated at 2×10⁵ cells/ml in 4 mlmedia in 6-well tissue culture plates and allowed to adhere for 1 hourat 37° C. Following cell adherence, 10 μg/ml (40 μg total) of eachpeptide were added to wells containing DCs, correlating to either 120 μgor 160 μg of total peptides added to each well (Campath-1H® 3-peptide or4-peptide groups), or 200 μg or 240 μg of total peptide added to eachwell (12G6-SFD1/K12 5-peptide or 6-peptide groups). 40 μg of the pan-DRbinding epitope (PADRE) were added to one well of DCs and served as apositive control, as it can bind to most HLA-DR molecules (Alexander J,et al., “Development of high potency universal DR-restricted helperepitopes by modification of high affinity DR-blocking peptides,”Immunity, 1:751-761 (1994)). Likewise, 40 μg of each of three HLA-DRbinding Tetanus toxoid peptides (DTIMMEPPYCKGLDIYYKA (SEQ ID NO: 183),SAMLTNLIIFGPGPVLNKNEV (SEQ ID NO: 184), and NNFTVSFWLRVPKVSASHLE (SEQ IDNO: 185)) were added to one well of DCs. Similarly, a heat inactivatedadenovirus was employed as a positive antigen source and was added toone well of DCs at 1 μg/ml. Lastly, one group of DCs remained unpulsedwith antigen and served as a ‘null’ educated group. The DCs pulsed withthe various antigens were incubated for at least three hours at 37° C.DCs were then treated with a ‘maturating cytokine cocktail’ containing50 ng/ml TNF-α, 10 ng/ml IL-6, 25 ng/ml IL-1beta (Peprotech, Rocky Hill,N.J.) and 500 ng/ml PGE-2 (Sigma Aldrich, St. Louis, Mo.). The antigenpulsed DCs were then allowed to mature overnight at 37° C.

Establishment of co-culture: Following peptide loading and maturation,DCs were washed twice with PBS and replenished with 4 ml RPMIsupplemented with 10% HS. Autologous CD4+ T cells were thawed andresuspended at 2×10⁶ cells/ml in RPMI supplemented with 10% HS,Penicillin, and Streptomycin. The DCs were then cultured with naïve CD4+T cells at a 10:1 T cell:DC ratio (8×10⁶ T cells:8×10⁵ DCs) in 8 mlsmedia. The co-culture was then incubated at 37° C. for 7 days.Approximately 72 hours after initiation of co-culture, the cells weresupplemented with 25 IU recombinant IL-2 (Peprotech, Rocky Hill, N.J.),and further supplemented with 25 IU recombinant IL-2 in fresh mediaevery 3-4 days thereafter.

Restimulation of co-culture: At day 7 (Stim #2) and day 14 (Stim #3),the co-cultures were restimulated following the above procedure.

Proliferation assay: DCs were plated, antigen pulsed and matured asstated above at 5×10⁵ cells/ml in 1 ml media on 24-well low bindingplates to ease the subsequent transfer of cells to U-bottom assayplates. An irrelevant HLA_DR binding peptide, CS 378-398 (peptidesequence DIEKKIAKMEKASSVFNVVNS (SEQ ID NO: 186)), was used as a negativecontrol (Alexander J, et al., “Development of high potency universalDR-restricted helper epitopes by modification of high affinityDR-blocking peptides,” Immunity, 1:751-761 (1994)). Following 24 hour DCmaturation, the cells were detached from plates using ice cold PBSwashes. DCs were plated in U-bottom 96 well plates with the antigenstimulated T cells at a 1:1 T cell:DC ratio (2.5×10⁴ DC/well). Each Tcell group was assayed in triplicate with DC pulsed with the educatingpeptide(s) (specific response) and DC pulsed with irrelevant peptide(nonspecific response), as well as T cell only and DC only controls. Theassay proceeded for 72 hours prior to the addition of 1 uCi tritiatedthymidine per well (Perkin Elmer, Waltham, Mass.). Cells were harvestedon a 96 well plate harvester (Perkin Elmer) and the amount of tritiatedthymidine incorporated quantified by measuring CPM on a Wallac MicrobetaTrilux counter (Perkin Elmer). The stimulation index was calculated bydividing the specific CPM by the nonspecific CPM.

T Cell Receptor (TCR) V beta usage: Any CD4+ T cells remaining afterestablishment of the proliferation assay were frozen for eventualdetermination of T cell receptor V beta chain expression. Cells werethawed and stained with antibodies recognizing 24 conjugated Vbetafamily members for 30 minutes following manufacturer's directions in theIOTest Beta Mark Kit (Beckman Coulter, France). After washing with PBSand resuspending in 1% formaldehyde, cells were analyzed on FACScalibur(Becton Dickinson, Franklin Lakes, N.J.). The percentage of cellsexpressing each of the detected V-beta chains was calculated, assummarized in FIG. 102 and FIG. 103.

Campath-1H® Immunogenicity Assessment

Immunogenicity assessment of Campath-1H® peptides was performed asdescribed above using PBMCs from ten normal donors, from BioMedSupply(BMS). The summary of the responses as indicated by the stimulationindex are depicted in Table 25A. Each donor is listed on one column, andeach row lists the group of peptides used to stimulate CD4+ T cells. TheStimulation index (SI) is determined by dividing the specific immuneresponse to the educating peptide group by an irrelevant response. SIvalues <2.0 are not listed. The proliferation data for each of the tendonors summarized in Table 25A is reported in FIG. 104A-J. Six donorsexhibited a stimulation index greater than 2.0, and as a result weretermed ‘Campath-1H® responders’. Educated CD4+ T cells from one of theresponders, BMS352, exhibited specific immune responses when assayedwith two different peptide groups. A seventh donor, BMS486, was alsoclassified as ‘responder’. In this donor, a stimulation index 1.7 timesbackground was recorded with the light chain peptide group 986-989. Whenassessing the V beta upregulation in the educated T cell cultures withinthis donor, it was shown that the 986-989 educated T cells exhibitedhigh upregulation of a single V beta, Vβ3 (FIG. 102). The upregulationof a single V beta and specific proliferative response indicated thatBMS486 was a Campath-1H® responder. The three non-responding donors,BMS200, BMS154, and BMS167, did not show proliferative data or V betaupregulation, indicating that a peptide specific immune response did notoccur. The Campath-1H® data was quantified as a 70% ( 7/10) responderrate. The total number of peptide groups eliciting an immune responsewas eight. Three of those eight immunogenic peptide groups elicitedstrong responses in the respective donors with stimulation indices of3.0 or above (Table 26).

Table 25: Summary of Stimulation Index Data

TABLE 25A Campath-1H ® Stimulation Index Campath-1H ® Stimulation IndexBMS200 BMS301 BMS154 BMS484 BMS362 BMS486 BMS150 BMS167 BMS170 BMS352982, 983, 984 nd & 987 988, 989 & 990 985, 986, 991 2.6 & 992 993, 994,995 & 996 997, 998 & 999 978, 979, 980, 981 982, 983, 984, 2.0 985 986,987, 988, 2.1 1.7 989 990, 991, 992, 993 994, 995, 996, 2.1 997 998,999, 1000, 1001 1002, 1003, 1004, 1005 1006, 1007, 4.2 5.4 1008, 10091010, 1011, 3.0 1012, 1013 1014, 1015, 1016, 1017 1018, 1019, 1020 PADRE2.0 2.0 2.5 2.5 2.8 10.5 2.6 Tetanus 11.2 2.3 4.5 2.3 25.9 Ad-Bgal-HI27.6 4.5 2.6 3.1 13.0 3.8 24.2 44.5 46.6 3.2 Null

TABLE 25B 12G6-SFD1/K12 Stimulation Index 12G6-SFD1/K12 StimulationIndex BMS484 BMS486 BMS656 BMS640 BMS361 BMS165 BMS902 BMS928 BMS927BMS963 1027, 1028, 1029, 1030, 1031 1032, 1072, 1073, 2.1 2.5 1074, 10361037, 1038, 1039, 1040, 1041 1042, 1043, 1075, 1076, 1077, 1078 1048,1049, 1050, 1079, 1080 1081, 1082, 1055, 2.1 1056, 1060 1061, 1062,1063, 2.0 nd 1064, 1065 1066, 1067, 1068, 2.0 1083, 1084, 1085 PADRE 3.03.6 2.2 9.8 2.4 2.3 4.7 TT-974, 975, 976 2.9 5.2 3.2 5.7 12.9 3.7 4.322.4 Ad-Bgal 17.6 11.0 10.9 31.7 29.1 29.3 10.3 8.4 6.3 Null

Example 70 Assessment of CD4+ T Cell Responses Induced by 12G6-SFD1/K12

Immunogenicity assessment and V beta analysis of the variable region of12G6-SFD1/K12 were performed as described in Example 69 for Campath-1H®,employing cells from ten normal donors. The proliferation data for eachof the ten donors summarized in Table 25B is reported in FIG. 105A-J.Two of these ten donors were also used in the Campath-1H® assessmentdescribed above (BMS486 and BMS484), while the remaining eight donorswere tested only with the 12G6-SFD1/K12 peptides. One donor, BMS484,responded to three peptide groups and was classified as a ‘12G6-SFD1/K12responder’ (Table 25B). Two donors, BMS927 and BMS928, each responded toone group of peptides and were therefore also classified as responders.Donor BMS928 showed a weak stimulation index of 2.0 to the groupcontaining heavy chain peptides 1066, 1067, 1068, 1083, 1084, and 1085.This response was confirmed by analyzing the proliferative T cells for Vbeta usage. The responding BMS928 T cells exhibited an upregulation of asingle V beta, Vβ20 (FIG. 103). Donor BMS927 showed a stimulation indexof 2.5 in T cells educated with one group of light chain peptides. Vbeta analysis of the responding BMS927 T cells did not indicate a singleV beta upregulation over background. However, this donor remains in the‘responder’ category, as the V beta kit represents only 70% of allpossible V beta usages. The 12G6-SFD1/K12 rate of immunogenicity inthese 10 donors was 30% ( 3/10), less than half the rate of Campath-1H®responders (70%). A total of five peptide groups elicited a response,while none of those five groups resulted in a stimulation index greaterthan 3.0 (Table 26).

TABLE 26 Summary of Campath-1H ® and 12G6-SFD1/K12 immune responses12G6- Campath-1H ® SFD1/K12 Percentage of responders 70% (7/10) 30%(3/10) Number of peptide groups eliciting 8 5 response Respondingpeptide groups with 3/8 (38%) 0/3 (0%) Stimulation Index ≧ 3.0

SUMMARY

Peptides correlating to the heavy and light chain variable regions ofhumanized anti-CD52 monoclonal antibody 12G6-SFD1/K12 induced fewerimmune responses from ten donors (30%) than peptides from the heavy andlight chain variable regions of Campath-1H® (70%). The CD4+ T cell basedimmune responses that were generated with 12G6-SFD1/K12 were also ofless magnitude than the Campath-1H® induced responses.

The following table lists the sequence identification numbers usedherein.

TABLE 26 List of SEQ ID NOs SEQ ID NO TYPE DESCRIPTION 1light chain variable Campath-1G ® 2 region (VL) CF1D12 3 8G3 (mouse) 44G7 (mouse) 5 9D9 (mouse) 6 11C11 (mouse) 7 3G7 (mouse) 8 5F7 (mouse) 912G6 (mouse) 10 23E6 (mouse) 11 2C3 (mouse) 12 7F11 (mouse) 134B10 (mouse) 14 heavy chain variable Campath-1G ® 15 region (VH) CF1D1216 8G3 (mouse) 17 4G7 (mouse) 18 9D9 (mouse) 19 11C11 (mouse) 203G7 (mouse) 21 5F7 (mouse) 22 12G6 (mouse) 23 23E6 (mouse) 242C3 (mouse) 25 7F11 (mouse) 26 4B10 (mouse) 27 light chain CDR-1Campath-1H ® 28 CF1D12 (mouse) 29 8G3, 4G7, 9D9, 11C11, 3G7 (mouse) 305F7 (mouse) 31 12G6, 23E6, 2C3 (mouse) 32 7F11 (mouse) 33 4B10 (mouse)34 light chain CDR-2 Campath-1H ® 35 CF1D12 (mouse) 368G3, 11C11, 12G6, 23E6, 2C3 (mouse) 37 4G7 (mouse) 38 9D9 (mouse) 393G7 (mouse) 40 5F7 (mouse) 41 7F11, 4B10 (mouse) 42 light chain CDR-3Campath-1H ® 43 CF1D12, 8G3, 4G7, 9D9, 11C11, 3G7, 5F7 (mouse) 4412G6 (mouse) 45 23E6 (mouse) 46 2C3 (mouse) 47 7F11 (mouse) 484B10 (mouse) 49 heavy chain CDR-1 Campath-1H ® 50CF1D12, 4G7, 9D9, 11C11, 3G7 (mouse) 51 8G3 (mouse) 52 5F7 (mouse) 5312G6 (mouse) 54 23E6 (mouse) 55 2C3 (mouse) 56 7F11, 4B10 (mouse) 57heavy chain CDR-2 Campath-1H ® 58 CF1D12 (mouse) 59 8G3 (mouse) 604G7 (mouse) 61 9D9, 11C11, 5F7 (mouse) 62 3G7 (mouse) 6312G6, 2C3 (mouse) 64 23E6 (mouse) 65 7F11 (mouse) 66 4B10 (mouse) 67heavy chain CDR-3 Campath-1H ® 68 CF1D12, 9D9 (mouse) 698G3, 4G7, 11C11, 3G7 (mouse) 70 5F7 (mouse) 71 12G6, 23E6 (mouse) 722C3 (mouse) 73 7F11 (mouse) 74 4B10 (mouse) 75 light chain primersLead-ML kappa (forward primer in leader sequence) 76FR1-ML kappa (forward primer in the framework 1) 77ML kappa const (reverse primer in constant region) 78VK-MK (forward primer in the framework 1) 79MKC-Const (reverse primer in constant region) 80 heavy chain primersMH-SP-ALT1 (forward primer in leader sequence) 81MH-SP-ALT2 (forward primer in leader sequence) 82MH-FR1 (forward primer in the framework 1) 83MH-FR1-1 (forward primer in the framework 1) 84MH-J2 (reverse primer in J region) 85MH-gamma-const (reverse primer in constant region) 86VH MH1 (forward primer in the framework 1) 87VH MH2 (forward primer in the framework 1) 88VH MH3 (forward primer in the framework 1) 89VH MH4 (forward primer in the framework 1) 90VH MH5 (forward primer in the framework 1) 91VH MH6 (forward primer in the framework 1) 92VH MH7 (forward primer in the framework 1) 93IgG1 (reverse primer in mouse IgG1 CH1 constant region) 94IgG2A (reverse primer in mouse IgG2A CH1 constant region) 95IgG2B (reverse primer in mouse IgG2B CH1 constant region) 96VH (partial) 4B10 (mouse): alignment 97 human germline (VH)VH3-72: alignment 98 VH (partial) 4B10 (humanized): alignment 99mouse VL (partial) 4B10 (mouse): alignment 100 human germline (VL)VK2-A18b: alignment 101 VL (partial) 4B10 (humanized): alignment 102 VL4B10-VK1 (humanized) 103 VH 4B10-VH1 (humanized) 104CD52 alanine-scanning WT 105 mutant peptides MUT 1 106 MUT 2 107 MUT 3108 MUT 4 109 MUT 5 110 MUT 6 111 MUT 7 112 MUT 8 113 MUT 9 114 MUT 10115 LC CDR-1 K/RSSQSLL/V/IXS/TN/DGXS/TYLX 116 K/RSSQSLL/V/IHS/TNGXS/TYLH117 RSSQSLVHTNGNS/TYLH 118 LC CDR-2 XVSXXXS 119 XVSXRXS 120 MVSXRFS 121LC CDR-3 XQXXH/R/KF/LN/IXX 122 SQSXH/R/KF/LN/IPX 123 SQSXHVPF/P 124HC CDR-1 GFXFXXYW/YMX 125 GFTFXXYW/YMX 126 GFTFTDYW/YMS 127 HC CDR-2XIRXKXBXYXTXYXXSVKG 128 XIRXKXNXYTTEYXXSVKG 129 FIRNKANGYTTEYXXSVKG 130HC CDR-3 TXXXY/F/W 131 TRYXY/F/WFDY 132 TRYIF/WFDY 133 JH6 WGQGTTVTVSS134 JK2 FGQGTKLEIK 135 JK5 FGQGTRLEIK 136 VH SFD1 7F11 137 SFD2 138 VLVK2 139 VH SFD1 2C3 140 12 141 15 142 16 143 17 144 19 145 VL VK1 146VK11 147 VK12 148 VK13 149 VH SFD1 12G6 150 VH10 151 VH11 152 VH12 153VL VK1 154 VK10 155 VK11 156 VK12 157 VK13 158 VH VH10 9D9 159 VH11 160VH15 161 VH16 162 VH17 163 VH18 164 VL VK2 165 VK12 166 VK13 167 VK14168 VK15 169 CD52 alanine-scanning MUT 1 170 peptides MUT 2 171 MUT 3172 MUT 4 173 MUT 5 174 MUT 6 175 MUT 7 176 MUT 8 177 MUT 9 178 MUT 10179 MUT 11 180 MUT 12 181 WT1 182 WT2 183 Tetanus toxoid HLA-DTIMMEPPYCKGLDIYYKA 184 DR-binding peptides SAMLTNLIIFGPGPVLNKNEV 185NNFTVSFWLRVPKVSASHLE 186 “irrelevant” HLA-DR- CS 378-398 binding peptide187 Campath-1H ® 978 188 LC overlapping 15-mer 979 189 peptides for 980190 immunogenicity study 981 191 982 192 983 193 984 194 985 195 986 196987 197 988 198 999 199 990 200 991 201 992 202 993 203 994 204 995 205996 206 997 207 Campath-1H ® 998 208 HC overlapping 15- 999 209mer peptides for 1000 210 immunogenicity study 1001 211 1002 212 1003213 1004 214 1005 215 1006 216 1007 217 1008 218 1009 219 1010 220 1011221 1012 222 1013 223 1014 224 1015 225 1016 226 1017 227 1018 228 1019229 1020 230 12G6-SFD1/K12 1027 231 LC 1028 232 overlapping 15-mer 1029233 peptides for 1030 234 immunogenicity study 1031 235 1032 236 1072237 1073 238 1074 239 1036 240 1037 241 1038 242 1039 243 1040 244 1041245 1042 246 1043 247 1075 248 1076 249 1077 250 1078 251 12G6-SFD1/K121048 252 HC 1049 253 overlapping 15-mer 1050 254 peptides for 1079 255immunogenicity study 1080 256 1081 257 1082 258 1055 259 1056 260 1060261 1061 262 1062 263 1063 264 1064 265 1065 266 1066 267 1067 268 1068269 1083 270 1084 271 1085 272 HC 2C3-SFD1 2C3 273 LC 2C3-K12 274 HC7F11-SFD1 7F11 275 LC 7F11-K2 276 HC 9D9-H16 9D9 277 9D9-H18 278 LC9D9-K13 279 HC 12G6-SFD1 12G6 280 LC 12G6-K12 281 HC 4B10-H1 4B10 282 LC4B10-K1 283 HC (nucleic acid) 2C3-SFD1 2C3 284 LC (nucleic acid) 2C3-K12285 HC (nucleic acid) 7F11-SFD1 7F11 286 LC (nucleic acid) 7F11-K2 287HC (nucleic acid) 9D9-H16 9D9 288 9D9-H18 289 LC (nucleic acid) 9D9-K13290 HC (nucleic acid) 12G6-SFD1 12G6 291 LC (nucleic acid) 12G6-K12 292HC (nucleic acid) 4B10-H1 4B10 293 LC (nucleic acid) 4B10-K1 294HC CDR-3 7F11-SFD2 (ARYIFFDY) 7F11

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A monoclonal anti-human CD52 antibody or anantigen-binding portion that binds to human CD52, wherein the lightchain and heavy chain of said antibody comprise the threecomplementarity determining regions (CDRs) found in a) SEQ ID NOs: 3 and16, respectively; b) SEQ ID NOs: 4 and 17, respectively; c) SEQ ID NOs:5 and 18, respectively; d) SEQ ID NOs: 6 and 19, respectively; e) SEQ IDNOs: 7 and 20, respectively; f) SEQ ID NOs: 8 and 21, respectively; g)SEQ ID NOs: 9 and 22, respectively; h) SEQ ID NOs: 10 and 23,respectively; i) SEQ ID NOs: 11 and 24, respectively; j) SEQ ID NOs: 12and 25, respectively; k) SEQ ID NOs: 12 and 137, respectively; or l) SEQID NOs: 13 and 26, respectively.
 2. A monoclonal anti-human CD52antibody or an antigen-binding portion that binds to human CD52, whereinthe heavy chain and the light chain of said antibody comprise the aminoacid sequences of SEQ ID NOs: 279 and 280, respectively, without thesignal sequences.
 3. A monoclonal anti-human CD52 antibody or anantigen-binding portion that binds to human CD52, wherein the lightchain of said antibody comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 102, 138, 145-148, 153-157, and164-168.
 4. A monoclonal anti-human CD52 antibody or an antigen-bindingportion that binds to human CD52, wherein the light chain of saidantibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 273, 275, 278, 280, and 282, without thesignal sequences.
 5. An antibody light chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 102, 138,145-148, 153-157, 164-168, 273, 275, 278, 280, and 282, without thesignal sequences if present.
 6. A monoclonal anti-human CD52 antibody oran antigen-binding portion that binds to human CD52, wherein the heavychain of said antibody comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 103, 136, 137, 139-144, 149-152, and158-163.
 7. A monoclonal anti-human CD52 antibody or an antigen-bindingportion that binds to human CD52, wherein the heavy chain of saidantibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 272, 274, 276, 277, 279, and 281, without thesignal sequences.
 8. An antibody heavy chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 103, 136,137, 139-144, 149-152, 158-163, 272, 274, 276, 277, 279, and 281,without the signal sequences if present.
 9. The monoclonal antibody orantigen-binding portion of claim 1, wherein the antibody is a humanizedantibody, a mouse antibody, or a chimeric antibody.
 10. The monoclonalantibody or antigen-binding portion of claim 1, wherein the frameworkregions of the heavy chain utilize a VH3-72 or VH3-23 human germlinesequence, and wherein the framework regions of the light chain utilize aVK2 A18b human germline sequence.
 11. The monoclonal antibody orantigen-binding portion of claim 1, wherein the antibody comprises heavychain (H)-CDR1, H-CDR2, H-CDR3, and light chain (L)-CDR1, L-CDR2, andL-CDR3 whose amino acid sequences are: a) SEQ ID NOs: 51, 59, 69, 29,36, and 43, respectively; b) SEQ ID NOs: 50, 60, 69, 29, 37, and 43,respectively; c) SEQ ID NOs: 50, 61, 68, 29, 38, and 43, respectively;d) SEQ ID NOs: 50, 61, 69, 29, 36, and 43, respectively; e) SEQ ID NOs:50, 62, 69, 29, 39, and 43, respectively; f) SEQ ID NOs: 52, 61, 70, 30,40, and 43, respectively; g) SEQ ID NOs: 53, 63, 71, 31, 36, and 44,respectively; h) SEQ ID NOs: 54, 64, 71, 31, 36, and 45, respectively;i) SEQ ID NOs: 55, 63, 72, 31, 36, and 46, respectively; j) SEQ ID NOs:56, 65, 73, 32, 41, and 47, respectively; k) SEQ ID NOs: 56, 65, 294,32, 41, and 47, respectively; or l) SEQ ID NOs: 56, 66, 74, 33, 41, and48, respectively.
 12. The monoclonal antibody or antigen-binding portionof claim 1, wherein the light chain and heavy chain of said antibodycomprise the amino acid sequences of: a) SEQ ID NOs: 3 and 16,respectively; b) SEQ ID NOs: 4 and 17, respectively; c) SEQ ID NOs: 5and 18, respectively; d) SEQ ID NOs: 6 and 19, respectively; e) SEQ IDNOs: 7 and 20, respectively; f) SEQ ID NOs: 8 and 21, respectively; g)SEQ ID NOs: 9 and 22, respectively; h) SEQ ID NOs: 10 and 23,respectively; i) SEQ ID NOs: 11 and 24, respectively; j) SEQ ID NOs: 12and 25, respectively; or k) SEQ ID NOs: 13 and 26, respectively.
 13. Themonoclonal antibody or antigen-binding portion of claim 1, wherein saidheavy chain and light chain comprise the amino acid sequences of a) SEQID NOs: 103 and 102, respectively; b) SEQ ID NOs: 136 and 138,respectively; c) SEQ ID NOs: 137 and 138, respectively; d) SEQ ID NOs:139 and 147, respectively; e) SEQ ID NOs: 149 and 155, respectively; f)SEQ ID NOs: 149 and 156, respectively; g) SEQ ID NOs: 158 and 165,respectively; h) SEQ ID NOs: 158 and 166, respectively; i) SEQ ID NOs:159 and 165, respectively; j) SEQ ID NOs: 159 and 166, respectively; k)SEQ ID NOs: 161 and 166, respectively; or l) SEQ ID NOs: 163 and 166,respectively.
 14. A monoclonal anti-human CD52 antibody or anantigen-binding portion that binds to human CD52, wherein the heavychain and the light chain of said antibody comprise the amino acidsequences of SEQ ID NOs: 272 and 273, respectively, without the signalsequences.
 15. A monoclonal anti-human CD52 antibody or anantigen-binding portion that binds to human CD52, wherein the heavychain and the light chain of said antibody comprise the amino acidsequences of SEQ ID NOs: 274 and 275, respectively, without the signalsequences.
 16. A monoclonal anti-human CD52 antibody or anantigen-binding portion that binds to human CD52, wherein the heavychain and the light chain of said antibody comprise the amino acidsequences of SEQ ID NOs: 276 and 278, respectively, without the signalsequences.
 17. A monoclonal anti-human CD52 antibody or anantigen-binding portion that binds to human CD52, wherein the heavychain and the light chain of said antibody comprise the amino acidsequences of SEQ ID NOs: 277 and 278, respectively, without the signalsequences.
 18. A monoclonal anti-human CD52 antibody or anantigen-binding portion that binds to human CD52, wherein the heavychain and the light chain of said antibody comprise the amino acidsequences of SEQ ID NOs: 281 and 282, respectively, without the signalsequences.
 19. The monoclonal antibody of any one of claims 1, 3, 6, and11-13, wherein said antibody is an IgG1, IgG2, IgG3, or IgG4 molecule.20. The antigen-binding portion of any one of claims 1, 3, and 6,wherein said portion is a single chain antibody, Fv, Fab, Fab′, F(ab′)₂,Fd, single chain Fv molecule (scFv), bispecific single chain Fv dimer,diabody, domain-deleted antibody or single domain antibody (dAb). 21.The monoclonal antibody or antigen-binding portion of claim 1, whereinsaid antibody or portion binds to an amino acid sequence comprising SEQID NO: 104, and optionally, the binding of said antibody or portion toSEQ ID NO: 104 is reduced by an alanine substitution at one or more ofresidues 4, 7, 8, and 11 of SEQ ID NO:
 104. 22. The monoclonal antibodyor antigen-binding portion of any one of claims 1, 3, and 6, whereinsaid antibody or antigen-binding portion has one or more propertiesselected from the group consisting of: a) depletes T or B lymphocytes,or both; b) preferentially depletes T lymphocytes as compared to Blymphocytes; c) increases circulating serum levels of TNF-alpha, IL-6,or MCP-1; d) mediates antibody-dependent cell mediated cytotoxicity(ADCC) of CD52-expressing cells; e) mediates complement-dependentcytotoxicity (CDC) of CD52-expressing cells; f) binds to human CD52 inthe presence of neutralizing antibodies to alemtuzumab; and g) promotesintracellular signaling in human T or B lymphocytes, or both.
 23. Acomposition comprising the monoclonal antibody or antigen-bindingportion of any one of claims 1, 2, 3, 4, 6, 7 and 11-18, and apharmaceutically acceptable vehicle or carrier.
 24. An isolated nucleicacid encoding the heavy chain, or the light chain, or both, of anantibody or antigen-binding portion of any one of claims 1, 2, 3, 4, 5,7, and 11-18.
 25. The isolated nucleic acid of claim 24, wherein saidisolated nucleic acid comprises: a) a heavy chain nucleotide sequenceselected from the group consisting of SEQ ID NOs: 283, 285, 287, 288,290, and 292, or said nucleotide sequence without the sequence encodinga signal peptide; b) a light chain nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 284, 286, 289, 291, and 293, or saidnucleotide sequence without the sequence encoding a signal peptide; orc) the nucleotide sequences of both a) and b).
 26. The isolated nucleicacid of claim 25, wherein said isolated nucleic acid comprises a heavychain nucleotide sequence and a light chain nucleotide sequence selectedfrom the group consisting of: a) SEQ ID NO: 283 and SEQ ID NO: 284,respectively, both without sequences encoding signal peptides; b) SEQ IDNO: 285 and SEQ ID NO: 286, respectively, both without sequencesencoding signal peptides; c) SEQ ID NO: 287 and SEQ ID NO: 289,respectively, both without sequences encoding signal peptides; d) SEQ IDNO: 288 and SEQ ID NO: 289, respectively, both without sequencesencoding signal peptides; e) SEQ ID NO: 290 and SEQ ID NO: 291,respectively, both without sequences encoding signal peptides; and f)SEQ ID NO: 292 and SEQ ID NO: 293, respectively, both without sequencesencoding signal peptides.
 27. A host cell comprising a first nucleicacid sequence encoding the heavy chain of a monoclonal antibody orantigen-binding portion of any one of claims 1, 2, 3, 4, 6, 7, and11-18, said first nucleic acid sequence operably linked to an expressioncontrol element, and a second nucleic acid sequence encoding the lightchain of said monoclonal antibody or antigen-binding portion, saidsecond nucleic acid sequence operably linked to an expression controlelement.
 28. A method of expressing an anti-human CD52 antibody or anantigen-binding portion that binds to human CD52, comprising maintainingthe host cell of claim 27 under conditions appropriate for expression ofthe antibody or portion, and expressing said antibody or portion.
 29. Amethod for inducing immunosuppression in a patient, comprisingadministering to the patient an effective amount of the antibody orantigen-binding portion of any one of claims 1, 2, 3, 4, 6, 7, and11-18.
 30. The method of claim 29, wherein the patient has an autoimmunedisease.
 31. A method for targeting CD52⁺cells in a cancer patient,comprising administering to the patient an effective amount of themonoclonal antibody or antigen-binding portion of any one of claims 1,2, 3, 4, 6, 7, and 11-18.
 32. A method of inhibiting angiogenesis in apatient in need thereof, comprising administering an effective amount ofthe monoclonal antibody or portion of any one of claims 1, 2, 3, 4, 6,7, and 11-18, to the patient.
 33. The method of claim 29, wherein thepatient is receiving transplantation.
 34. The method of claim 30,wherein said autoimmune disease is multiple sclerosis, rheumatoidarthritis, systemic lupus erythematosus, or vasculitis.
 35. The methodof claim 31, wherein the cancer patient has leukemia.
 36. The method ofclaim 31, wherein the cancer patient has lymphoma.
 37. The method ofclaim 31, wherein the cancer patient has T cell malignancy, and theantibody or portion preferentially depletes T cells as compared to Bcells.
 38. The method of claim 31, wherein the cancer patient has asolid tumor.
 39. The method of claim 29, further comprisingadministering to the patient a neutrophil or NK cell stimulatory agent.40. The method of claim 31, further comprising administering to thepatient a neutrophil or NK cell stimulatory agent.
 41. The method ofclaim 29, further comprising administering to the patient a T regulatorycell stimulatory agent.
 42. The method of claim 31, further comprisingadministering to the patient a T regulatory cell stimulatory agent. 43.The method of claim 32, wherein the patient has a solid tumor.
 44. Themethod of claim 32, wherein the patient has neovascularization.